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.
