Name: analysis of scap n-glycosylation and trafficking in human cells
Uploaded: 2016-11-08T14:00:00
Description: Watch this Scientific Journal Video about Analysis of SCAP N-glycosylation and Trafficking in Human Cells at JoVE.com

We are grateful to Drs. Mike S. Brown and Joseph L. Goldstein for their agreement to publish this method according to the methods described in their publications. We appreciate Dr. Peter Espenshade for sharing the GFP-SCAP plasmid. This work was supported by NIH grants NS072838 and NS079701 to D.G., American Cancer Society Research Scholar Grant RSG-14-228-01-CSM to D.G., and OSUCCC Pelotonia Postdoctoral Fellowship to C.C. We also appreciate the support from the Ohio State Neuroscience Core (P30 NS038526) and OSUCCC Translational Therapeutic Program seed grant and start-up funds to D.G.

1. Detection of Endogenous SCAP Protein in Human Cells
<ol>
	<li>Cell Culture and Treatment
	<ol>
		<li>Seed ~ 1 &#215; 106 U87 cells in a 10 cm dish with Dulbecco&#39;s Modified Eagle Medium (DMEM) supplemented with 5% fetal bovine serum (FBS) and incubate the cells at 37 &#176;C and 5% CO2 for 24 hr before treatment.</li>
		<li>Wash cells once with phosphate-buffered saline (PBS), then switch cells to fresh DMEM medium with or without glucose (5 mM) for 12 hr.</li>
	</ol>
	</li>
	<li>Preparation...

<ol>
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L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulation+of+sterol+synthesis+in+eukaryotes.">Regulation of sterol synthesis in eukaryotes.</a> Annu Rev Genet. 41, 401-427 (2007).</li><li>Yang, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Crucial+step+in+cholesterol+homeostasis:+sterols+promote+binding+of+SCAP+to+INSIG-1,+a+membrane+protein+that+facilitates+retention+of+SREBPs+in+ER.">Crucial step in cholesterol homeostasis: sterols promote binding of SCAP to INSIG-1, a membrane protein that facilitates retention of SREBPs in ER.</a> Cell. 110, 489-500 (2002).</li><li>Yabe, D., Brown, M. S., Goldstein, J. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Insig-2,+a+second+endoplasmic+reticulum+protein+that+binds+SCAP+and+blocks+export+of+sterol+regulatory+element-binding+proteins.">Insig-2, a second endoplasmic reticulum protein that binds SCAP and blocks export of sterol regulatory element-binding proteins.</a> Proc Natl Acad Sci U S A. 99, 12753-12758 (2002).</li><li>Yabe, D., Xia, Z. P., Adams, C. M., Rawson, R. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Three+mutations+in+sterol-sensing+domain+of+SCAP+block+interaction+with+insig+and+render+SREBP+cleavage+insensitive+to+sterols.">Three mutations in sterol-sensing domain of SCAP block interaction with insig and render SREBP cleavage insensitive to sterols.</a> Proc Natl Acad Sci U S A. 99, 16672-16677 (2002).</li><li>Espenshade, P. J., Li, W. 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J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulated+step+in+cholesterol+feedback+localized+to+budding+of+SCAP+from+ER+membranes.">Regulated step in cholesterol feedback localized to budding of SCAP from ER membranes.</a> Cell. 102, 315-323 (2000).</li><li>Sakai, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sterol-regulated+release+of+SREBP-2+from+cell+membranes+requires+two+sequential+cleavages,+one+within+a+transmembrane+segment.">Sterol-regulated release of SREBP-2 from cell membranes requires two sequential cleavages, one within a transmembrane segment.</a> Cell. 85, 1037-1046 (1996).</li><li>Wang, X., Sato, R., Brown, M. S., Hua, X., Goldstein, J. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=SREBP-1,+a+membrane-bound+transcription+factor+released+by+sterol-regulated+proteolysis.">SREBP-1, a membrane-bound transcription factor released by sterol-regulated proteolysis.</a> Cell. 77, 53-62 (1994).</li><li>Brown, M. S., Goldstein, J. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+proteolytic+pathway+that+controls+the+cholesterol+content+of+membranes,+cells,+and+blood.">A proteolytic pathway that controls the cholesterol content of membranes, cells, and blood.</a> Proceedings of the National Academy of Sciences of the United States of America. 96, 11041-11048 (1999).</li><li>Rawson, R. B., DeBose-Boyd, R., Goldstein, J. L., Brown, M. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Failure+to+cleave+sterol+regulatory+element-binding+proteins+(SREBPs)+causes+cholesterol+auxotrophy+in+Chinese+hamster+ovary+cells+with+genetic+absence+of+SREBP+cleavage-activating+protein.">Failure to cleave sterol regulatory element-binding proteins (SREBPs) causes cholesterol auxotrophy in Chinese hamster ovary cells with genetic absence of SREBP cleavage-activating protein.</a> J Biol Chem. 274, 28549-28556 (1999).</li><li>Zelenski, N. G., Rawson, R. B., Brown, M. S., Goldstein, J. 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S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Autocatalytic+processing+of+site-1+protease+removes+propeptide+and+permits+cleavage+of+sterol+regulatory+element-binding+proteins.">Autocatalytic processing of site-1 protease removes propeptide and permits cleavage of sterol regulatory element-binding proteins.</a> The Journal of biological chemistry. 274, 22795-22804 (1999).</li><li>Guo, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=SCAP+links+glucose+to+lipid+metabolism+in+cancer+cells.">SCAP links glucose to lipid metabolism in cancer cells.</a> Mol Cell Oncol. 3, (2016).</li><li>Cheng, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Glucose-Mediated+N-glycosylation+of+SCAP+Is+Essential+for+SREBP-1+Activation+and+Tumor+Growth.">Glucose-Mediated N-glycosylation of SCAP Is Essential for SREBP-1 Activation and Tumor Growth.</a> Cancer Cell. 28, 569-581 (2015).</li><li>Shao, W., Espenshade, P. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sugar+Makes+Fat+by+Talking+to+SCAP.">Sugar Makes Fat by Talking to SCAP.</a> Cancer Cell. 28, 548-549 (2015).</li><li>Guo, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=EGFR+signaling+through+an+Akt-SREBP-1-dependent,+rapamycin-resistant+pathway+sensitizes+glioblastomas+to+antilipogenic+therapy.">EGFR signaling through an Akt-SREBP-1-dependent, rapamycin-resistant pathway sensitizes glioblastomas to antilipogenic therapy.</a> Sci Signal. 2, 82 (2009).</li><li>Guo, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+LXR+agonist+promotes+GBM+cell+death+through+inhibition+of+an+EGFR/AKT/SREBP-1/LDLR-dependent+pathway.">An LXR agonist promotes GBM cell death through inhibition of an EGFR/AKT/SREBP-1/LDLR-dependent pathway.</a> Cancer Discov. 1, 442-456 (2011).</li><li>Guo, D., Bell, E. 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In this study, we describe a protocol for the isolation of membrane fractions in human cells and for the preparation of the samples for the detection of SCAP N-glycosylation and total protein by using western blot. We further provide a method to monitor SCAP trafficking by using GFP-labeling and confocal microscopy. The method is specifically used to analyze membrane protein and is an important tool to investigate SCAP N-glycosylation and trafficking.
Compared with the method...

Deregulation of lipid metabolism is a common characteristic of cancer and metabolic diseases1-6. In these processes, sterol regulatory element-binding proteins (SREBPs), a family of transcription factors, play a critical role in controlling the expression of genes important for the uptake and synthesis of cholesterol, fatty acids, and phospholipids7-10.
SREBPs, including SREBP-1a, SREBP-1c, and SREBP-2, are synthesized as inactive precursors that bind to the endoplasmic reticulum (ER) membrane by virtue of two transmembrane domains7. The N-terminus of SREBPs contains a DNA-binding domain for the transactivation of target genes11. The C-terminus of SREBP precursors binds to SREBP cleavage-activating protein (SCAP)12,13, a polytopic membrane protein that plays a pivotal role in the regulation of SREBP stability and activation14-16.
The implementation of transcriptional function requires the translocation of the SCAP/SREBP complex from the ER to the Golgi, where two proteases sequentially cleave SREBP and release its N-terminal fragment, which then enters into the nucleus for the transactivation of lipogenic genes7. In these processes, the levels of sterols in the ER membrane control the exit of the SCAP/SREBP complex from the ER7,17. Under high sterol conditions, sterol binds to SCAP or ER-resident insulin-induced gene protein-1 (Insig-1) or -2 (Insig-2), enhancing the association of SCAP with Insigs that retain the SCAP/SREBP complex in the ER18-20. When sterol levels decrease, SCAP dissociates with Insigs. This leads to a SCAP conformational change, which allows SCAP interaction with common coat proteins (COP)II complex. The complex mediates the incorporation of the SCAP/SREBP complex into the budding vesicles and directs its transport from the ER to the Golgi21,22. Upon translocation to the Golgi, SREBPs are sequentially cleaved by Site-1 and Site-2 proteases, leading to the release of the N-terminus7,23-29.
SCAP protein carries three N-linked oligosaccharides at asparagine (N) positions N263, N590, and N64115. We recently revealed that glucose-mediated N-glycosylation of SCAP in these sites is a prerequisite condition for SCAP/SREBP trafficking from the ER to the Golgi under low sterol conditions30-32. Loss of SCAP glycosylation via mutation of all three asparagine to glutamine (NNN to QQQ) disables the trafficking of the SCAP/SREBP complex and results in the instability of SCAP protein and reduction of SREBP activation31. Our recent data also demonstrate that SREBP-1 is highly activated in glioblastoma and regulated by SCAP N-glycosylation4,31,33,34. Targeting SCAP/SREBP-1 signaling is emerging as a new strategy to treat malignancies and metabolic syndromes1,3,35-38. Therefore, it is important to develop an effective method to analyze SCAP protein and N-glycosylation levels and monitor its trafficking in human cells and patient tissues.
The luminal region of SCAP protein (amino acids 540-707) in the ER contains two N-glycosylation sites (N590 and N641) that are protected from proteolysis when intact membranes are treated with trypsin15. This luminal fragment has a molecular weight of ~ 30 kDa that is small enough to allow the resolution of individual glycosylated variants of SCAP by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE)15,31. Here, we provide a method to detect N-glycosylation of SCAP and the total protein in human cells. This protocol is derived from the method described in the publications from the Brown and Goldstein laboratory15 and our recent publication31. The protocol can be used in the study of SCAP protein from mammalian cells.

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			<td height="21" style="height:21px;width:259px;">X-treme GENE HP DNA Transfection Reagent&#160;</td>
			<td style="width:163px;">Roche</td>
			<td style="width:123px;">6366236001</td>
			<td style="width:91px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Opti-MEMI medium&#160;</td>
			<td>Life Technologies</td>
			<td>31985-070</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">trypsin&#160;</td>
			<td>Sigma</td>
			<td>T6567</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">soybean trypsin inhibitor</td>
			<td>Sigma</td>
			<td>T9777</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">PNGase F&#160;</td>
			<td>Sigma</td>
			<td>P7367</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Anti-SCAP (9D5) antibody&#160;&#160;</td>
			<td>Santa Cruz</td>
			<td>sc-13553</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">GFP antibody&#160;</td>
			<td>Roche</td>
			<td>11814460001</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">SREBP-1 antibody (IgG-2A4)</td>
			<td>BD Pharmingen</td>
			<td>557036</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">PDI Antibody (H-17)</td>
			<td>Santa Cruz</td>
			<td style="width:123px;">sc-30932</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Lamin A Antibody (H-102)</td>
			<td>Santa Cruz</td>
			<td style="width:123px;">sc-20680</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">SCAP antibody (a.a 450-500)</td>
			<td>Bethyl Laboratories, Inc.</td>
			<td>A303-554A</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Dulbecco&#8217;s modified Eagle&#8217;s medium (DMEM) without glucose, pyruvate and glutamine&#160;&#160;</td>
			<td>Cellgro</td>
			<td>17-207-CV</td>
			<td>Add 1 Mm Pyravate and 4 mM Glutamine before use</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">22G x 1 1/2 needle&#160;</td>
			<td>BD&#160;</td>
			<td>305156</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">pepstatin A</td>
			<td>Sigma</td>
			<td>P5318</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">leupeptin</td>
			<td>Sigma</td>
			<td>L2884</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">PMSF</td>
			<td>Sigma</td>
			<td>P7626</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">DTT</td>
			<td>Sigma</td>
			<td>43819</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">ALLN</td>
			<td>Sigma</td>
			<td>A6185</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Nitrocellulose membrane</td>
			<td>GE</td>
			<td>RPN3032D</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">EDTA Solution 0.5 M PH8.5 100 ml&#160;</td>
			<td>VWR</td>
			<td>82023-102&#160;</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">EGTA 0.5 M sterile (PH 8.0) 50 ml</td>
			<td>Fisher Scentific</td>
			<td>50255956</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">HyClone FBS</td>
			<td>Thermo scientific</td>
			<td>SH3007103</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">glycerol</td>
			<td>Sigma</td>
			<td>G5516</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">&#946;-mercaptoethanol&#160;</td>
			<td>Sigma</td>
			<td>M3148</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">bromophenol blue</td>
			<td>Sigma</td>
			<td>B8026</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">prolong gold antifade reagent with dapi</td>
			<td>life technologies</td>
			<td>P36935</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">L-glutamine (200 mM)</td>
			<td>life technologies</td>
			<td>25030081</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">sodium pyruvate</td>
			<td>life technologies</td>
			<td>11360-070</td>
			<td></td>
		</tr>
	</tbody>
</table>

analysis of scap n-glycosylation and trafficking in human cells

Elevated lipogenesis is a common characteristic of cancer and metabolic diseases. Sterol regulatory element-binding proteins (SREBPs), a family of membrane-bound transcription factors controlling the expression of genes important for the synthesis of cholesterol, fatty acids and phospholipids, are frequently upregulated in these diseases. In the process of SREBP nuclear translocation, SREBP-cleavage activating protein (SCAP) plays a central role in the trafficking of SREBP from the endoplasmic reticulum (ER) to the Golgi and in subsequent proteolysis activation. Recently, we uncovered that glucose-mediated N-glycosylation of SCAP is a prerequisite condition for the exit of SCAP/SREBP from the ER and movement to the Golgi. N-glycosylation stabilizes SCAP and directs SCAP/SREBP trafficking. Here, we describe a protocol for the isolation of membrane fractions in human cells and for the preparation of the samples for the detection of SCAP N-glycosylation and total protein by using western blot. We further provide a method to monitor SCAP trafficking by using confocal microscopy. This protocol is appropriate for the investigation of SCAP N-glycosylation and trafficking in mammalian cells.

Figure 1 shows the detection of endogenous SCAP protein and SREBP-1 nuclear form in human glioblastoma U87 cells in response to glucose stimulation by using western blot. The membrane protein SCAP is detected in the &#34;membrane fraction&#34;. The nucleus form (N-terminal) of SREBP-1 is detected in the &#34;nuclear extracts&#34;. The level of SCAP protein (PDI serves as an internal control of ER membrane proteins) and SREBP-1 nuclear form are markedly enhanced by ...

Watch this Scientific Journal Video about Analysis of SCAP N-glycosylation and Trafficking in Human Cells at JoVE.com

Analysis of SCAP N-glycosylation and Trafficking in Human Cells

We describe a modified method for membrane fraction isolation from human cells and sample preparation for the detection of SCAP N-glycosylation and total protein by using western blot. We further introduce a GFP-labeling method to monitor SCAP trafficking using confocal microscopy. This protocol can be used in regular biology laboratories.

Analysis of SCAP N-glycosylation and Trafficking in Human Cells

Elevated lipogenesis is a common characteristic of cancer and metabolic diseases. Sterol regulatory element-binding proteins (SREBPs), a family of ...

The overall goal of this protocol is to provide a simple approach of analyzing SCAP N-glycosylation and its trafficking in human cells. This method is used to analyze the N-glycosylation of SCAP, which is indispensable for SCAP trafficking and activation of SREBP-mediated lipid metabolism in mammalian cells. Compare with the conventional method use lectins, our method provides a simple procedure in identifying SCAP N-glycosylation, will detecting its migration shift after removing N-glycan use PNGase.Though this method can provide an insight into cancer, it can also be applied to other systems, such as cardiovascular diseases and metabolic syndromes. To begin this procedure, seed one times 10 to the six U87 cells in a 10-centimeter dish with DMEM supplemented with 5%FBS, and incubate the cells at 37 degrees Celsius and 5%CO2 for 24 hours before treatment. Next, wash the cells once with PBS.Then, switch the PBS to fresh DMEM medium with or without glucose for 12 hours. To prepare cell membrane fractions and nuclear extracts, wash the cells once with PBS. Next, scrape the cells into one milliliter of PBS, and centrifuge at 1, 000 times g for five minutes at four degrees Celsius.Afterward, resuspend the cells in ice-cold buffer on ice for 30 minutes. Subsequently, pass the cell extracts through a 22-gauge needle 30 times, and centrifuge at 890 times g at four degrees Celsius for five minutes to isolate the nuclei. Then, use the supernatant for the separation of membrane fractions, and resuspend the nuclear pellet in 0.1 milliliters of buffer C.Following this, rotate the suspension at four degrees Celsius for 60 minutes, and centrifuge it at 20, 000 times g for 20 minutes at four degrees Celsius.Subsequently, heat the nuclear extracts at 100 degrees Celsius for 10 minutes with 5x loading buffer before subjecting them to SDS-PAGE. Afterward, centrifuge the supernatant at 20, 000 times g for 20 minutes at four degrees Celsius. For western blot analysis, dissolve the pellet in 0.1 milliliters of SDS lysis buffer.Then, incubate the membrane fraction at 37 degrees Celsius for 30 minutes, and determine the protein concentration by the Bradford protein assay. Next, add one microliter of 100x bromophenol blue solution before subjecting the sample to SDS-PAGE. Load 25 to 50 micrograms of the total membrane proteins on a 10%SDS-PAGE gel.Subsequently, run the gel at 80 volts for 15 minutes. Then, change to 130 volts, and run for another 115 minutes. Transfer the proteins from the SDS-PAGE gel to a nitrocellulose membrane at 140 milliamps for 130 minutes.For western blotting analysis, use anti-SCAP antibody to detect the total SCAP protein and PDI, an ER-resident protein, as an internal control. In addition, use anti-SREBP-1 antibody to detect the N-terminal band of SREBP-1, and use lamin A as an internal control for nuclear extracts. For membrane analysis, seed one times 10 to the six HEK293T cells in a 10-centimeter dish with DMEM supplemented with 5%FBS, and incubate the cells at 37 degrees Celsius and 5%CO2 for 24 hours before transfection.Next, dilute four to eight micrograms of GFP-SCAP plasmid in one milliliter of the reduced serum medium, and mix gently. Then, add eight to 16 microliters of DNA transfection reagent by pipetting it directly into the reduced serum medium containing plasmids, and mix gently. Incubate the transfection reagent and plasmid complex for 25 minutes at room temperature.Afterward, add the transfection complex to the cells with fresh medium, and continue to culture for 24 hours at 5%CO2 and 37 degrees Celsius. Wash them once with PBS, and treat them for 12 hours with or without glucose in the presence or absence of tunicamycin, an N-glycosylation inhibitor. Next, prepare the cell membrane pellets from the cells overexpressing GFP-SCAP according to the accompanying manuscript.For trypsin proteolysis, resuspend the cell membrane pellets in 114 microliters of buffer. Incubate 57 microliters of membrane proteins in the absence or presence of one microliter of trypsin for 30 minutes at 30 degrees Celsius. After 30 minutes, stop the reactions by adding two microliters of soybean trypsin inhibitor.For the deglycosylation with PNGase F, add 10 microliters of solution containing SDS and 2-mercaptoethanol to the sample. Then, heat the mixture at 100 degrees Celsius for 10 minutes. After that, sequentially add sodium phosphate, Nonidet P-40 with protease inhibitors, and PNGase F, and incubate the sample at 37 degrees Celsius for three hours.Then, stop the reactions by adding 5x loading buffer, and heat the mixture at 100 degrees Celsius for 10 minutes. Subsequently, load 25 to 50 micrograms of total membrane proteins on a 10%SDS-PAGE gel. Run the gel at 80 volts for 15 minutes.Then, change to 130 volts for another 115 minutes, followed by 140 milliamps for 130 minutes. Transfer the proteins from the SDS-PAGE gel to a nitrocellulose membrane at 140 milliamps for 130 minutes. For western blot, use 10 micrograms per milliliter of anti-SCAP antibody to detect N-glycosylation and the total SCAP protein.In this procedure, seed 2.5 times 10 to the five U87 cells in a 60-millimeter dish with DMEM supplemented with 5%FBS. Incubate the cells at 37 degrees Celsius and 5%CO2 for 24 hours before transfection. Then, dilute four micrograms of GFP-SCAP plasmid in 0.5 milliliters of reduced serum medium.Next, add eight microliters of DNA transfection reagent by pipetting it directly into the reduced serum medium containing plasmids, and mix gently. After that, incubate the transfection reagent and plasmid complex for 25 minutes at room temperature. Add the transfection complex to the cells with fresh medium, and continue to culture for 24 hours at 5%CO2 and 37 degrees Celsius.Reseed five to 10 times 10 to the four cells into a six-well plate containing a glass coverslip, and continue to culture at 37 degrees Celsius and 5%CO2 for 24 hours. Wash the cells once with PBS, and treat them for 12 hours with or without glucose in the presence or absence of tunicamycin. Then, wash the cells once with PBS, and fix them in 4%formaldehyde for 10 minutes.Afterward, wash the cells three times with PBS. Invert the coverslips, mount them on slides together with antifade reagent, and seal the coverslips using nail polish. Following this, check the GFP-SCAP trafficking using a 63x oil-immersion objective in a laser-scanning confocal microscope.Western blotting shows that glucose stimulation markedly enhanced the levels of SCAP protein and SREBP-1 nuclear formed in human glioblastoma U87 cells. The higher weight bands in this figure indicate SCAP protein containing one or two N-linked oligosaccharides in the presence of glucose and absence of PNGase or tunicamycin. In the presence of PNGase F, N-linked oligosaccharides were removed from SCAP protein, leading to the fragment moving faster in the SDS-PAGE.Inconsistent, blocking the N-glycosylation initiation process by tunicamycin completely abolished SCAP N-glycosylation, which was shown by the appearance of a lower band of SCAP fragment. This figure shows that GFP-SCAP trafficking from the ER to the Golgi in response to glucose stimulation was suppressed by tunicamycin treatment in U87 cells. These confocal microscopy images indicate that GFP-SCAP protein resides in the ER in the absence of glucose, as shown by its co-localization with PDI.In contrast, in the presence of glucose, GFP-SCAP moves to the Golgi, shown by the co-localization with the Golgi protein marker giantin. Moreover, tunicamycin treatment suppressed glucose-induced trafficking of GFP-SCAP from the ER to the Golgi. After its development, this technique paved the way for researchers in the field of lipid metabolism to explore metabolic alterations in human patients with cancer or metabolic syndromes.After watching this video, you should have a good understanding of how to detect SCAP N-glycosylation and analyze its trafficking in human cells.

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