Name: isolation of whole cell protein lysates from mouse facial processes and cultured palatal mesenchyme cells for phosphoprotein analysis
Uploaded: 2022-04-01T15:00:00
Description: Watch this Scientific Journal Video about Isolation of Whole Cell Protein Lysates from Mouse Facial Processes and Cultured Palatal Mesenchyme Cells for Phosphoprotein Analysis at JoVE.com

129S4 mice were a gift from Dr. Philippe Soriano, Icahn School of Medicine at Mount Sinai. This work was supported with funds from the National Institutes of Health (NIH)/National Institute of Dental and Craniofacial Research (NIDCR) R01 DE027689 and K02 DE028572 to K.A.F., F31 DE029976 to M.A.R. and F31 DE029364 to B.J.C.D.

All the procedures involving animals were approved by the Institutional Animal Care and Use Committee (IACUC) of the University of Colorado Anschutz Medical Campus and performed in compliance with institutional guidelines and regulations. Female 129S4 mice at 1.5-6 months of age and housed at a sub-thermoneutral temperature of 21-23 &#176;C were used for embryo harvests. A schematic workflow of the protocol is represented in Figure 1. See the Table of Materials for details r...

The protocol described here allows researchers to probe critical phosphorylation-dependent signaling events during craniofacial development in a robust and reproducible manner. There are several critical steps in this protocol that ensure proper collection of data and analysis of results. Whether isolating phosphoproteins from mouse facial processes and/or cultured palatal mesenchyme cells, it is imperative to move quickly and efficiently while keeping all reagents and materials on ice when indicated. The low temperature...

Mammalian craniofacial development is a complex morphological process during which multiple cell populations coordinate to generate the frontonasal skeleton. In the mouse, this process begins at embryonic day (E) 9.5 with the formation of the frontonasal prominence and pairs of maxillary and mandibular processes, each of which contains post-migratory cranial neural crest cells. The lateral and medial nasal processes arise from the frontonasal prominence with the appearance of the nasal pits and eventually fuse to form the nostrils. Further, the medial nasal processes and maxillary processes fuse to generate the upper lip. Concurrently, palatogenesis is initiated with the formation of distinct outgrowths - the secondary palatal shelves - from the oral side of the maxillary processes at E11.5. Over time, the palatal shelves grow downward on either side of the tongue, elevate to an opposing position above the tongue, and eventually fuse at the midline to form a continuous palate that separates the nasal and oral cavities by E16.51.
These morphological changes throughout craniofacial development are initiated and sustained through diverse signaling interactions, which often include protein phosphorylation by kinases. For example, cell membrane receptors, such as subfamilies of transforming growth factor (TGF)-&#946; receptors, including bone morphogenetic protein receptors (BMPRs), and various receptor tyrosine kinase (RTK) families, are autophosphorylated upon ligand binding and activation in cranial neural crest cells2,3,4. Additionally, the G protein-coupled transmembrane receptor Smoothened becomes phosphorylated in cranial neural crest cells and craniofacial ectoderm downstream of Sonic hedgehog (SHH) ligand binding to the Patched1 receptor, resulting in Smoothened accumulation at the ciliary membrane and SHH pathway activation5. Such ligand-receptor interactions can occur through autocrine, paracrine, and/or juxtacrine signaling in craniofacial contexts. For example, BMP6 is known to signal in an autocrine manner during chondrocyte differentiation6, whereas fibroblast growth factor (FGF) 8 is expressed in the pharyngeal arch ectoderm and binds to members of the FGF family of RTKs expressed in the pharyngeal arch mesenchyme in a paracrine fashion to initiate patterning and outgrowth of the pharyngeal arches7,8,9,10. Furthermore, Notch signaling is activated in both chondrocytes and osteoblasts during craniofacial skeletal development through juxtacrine signaling when transmembrane Delta and/or Jagged ligands bind to transmembrane Notch receptors on neighboring cells, which are subsequently cleaved and phosphorylated11. However, there are other ligand and receptor pairs important for craniofacial development that have the flexibility to function in both autocrine and paracrine signaling. As an example, during murine tooth morphogenesis, platelet-derived growth factor (PDGF)-AA ligand has been demonstrated to signal in an autocrine manner to activate the RTK PDGFR&#945; in the enamel organ epithelium12. In contrast, in murine facial processes during mid-gestation, transcripts encoding the ligands PDGF-AA and PDGF-CC are expressed in the craniofacial ectoderm, while the PDGFR&#945; receptor is expressed in the underlying cranial neural crest-derived mesenchyme, resulting in paracrine signaling13,14,15,16,17. Regardless of the signaling mechanism, these receptor phosphorylation events often result in the recruitment of adaptor proteins and/or signaling molecules, which frequently become phosphorylated themselves to initiate intracellular kinase cascades such as the mitogen-activated protein kinase (MAPK) pathway18,19.
The terminal intracellular effectors of these cascades can then phosphorylate an array of substrates, such as transcription factors, RNA-binding, cytoskeletal and extracellular matrix proteins. Runx220, Hand121, Dlx3/522,23,24, Gli1-325, and Sox926 are among the transcription factors phosphorylated in the context of craniofacial development. This post-translational modification (PTM) can directly affect susceptibility to alternative PTMs, dimerization, stability, cleavage, and/or DNA-binding affinity, among other activities20,21,25,26. Additionally, the RNA-binding protein Srsf3 is phosphorylated in the context of craniofacial development, leading to its nuclear translocation27. In general, phosphorylation of RNA-binding proteins has been shown to affect their subcellular localization, protein-protein interactions, RNA binding, and/or sequence specificity28. Furthermore, phosphorylation of actomyosin can lead to cytoskeletal rearrangements throughout craniofacial development29,30, and phosphorylation of extracellular matrix proteins, such as small integrin-binding ligand N-linked glycoproteins, contributes to biomineralization during skeletal development31. Through the above and numerous other examples, it is evident that there are wide implications for protein phosphorylation during craniofacial development. Adding an additional level of regulation, protein phosphorylation is further modulated by phosphatases, which counteract kinases by removing phosphate groups.
These phosphorylation events at both the receptor and effector molecule levels are critical for the propagation of signaling pathways and ultimately result in changes in gene expression in the nucleus, driving specific cell activities, such as migration, proliferation, survival, and differentiation, which result in proper formation of the mammalian face. Given the context specificity of protein interactions with kinases and phosphatases, the resulting changes in PTMs, and their effects on cell activity, it is critical that these parameters be studied in a physiologically-relevant setting to gain complete understanding of the contribution of phosphorylation events to craniofacial development. Here, examples of two contexts in which to study phosphorylation of proteins and, thus, activation of signaling pathways during mammalian craniofacial development are provided: mouse facial processes, in particular E11.5 maxillary processes, and cultured mouse embryonic palatal mesenchyme cells derived from E13.5 secondary palatal shelves - both primary32 and immortalized33. At E11.5, the maxillary processes are in the process of fusing with the lateral and medial nasal processes1, thereby representing a critical timepoint during mouse craniofacial development. Further, maxillary processes and cells derived from the palatal shelves were chosen here because the latter structures are derivatives of the former, thereby providing researchers the opportunity to interrogate protein phosphorylation in vivo and in vitro in related contexts. However, this protocol is also applicable to alternative facial processes and developmental timepoints.
A critical problem in studying phosphorylated proteins is that they are easily dephosphorylated during protein isolation by abundant environmental phosphatases. To overcome this barrier, adaptations and modifications to standard laboratory methods that allow for isolation of phosphorylated proteins are discussed. Additionally, best practices are provided for proper analysis and quantification of phosphorylated proteins. These techniques, particularly in combination with pharmacological inhibitors and/or murine genetic models, can be used to gain greater insight into the dynamics and roles of various signaling pathways active during craniofacial development.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Equipment</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Block for mini dry bath</td>
			<td>Research Products International Corp</td>
			<td>400783</td>
			<td></td>
		</tr>
		<tr>
			<td>ChemiDoc XRS+ imaging system with Image Lab software</td>
			<td>Bio-Rad</td>
			<td>1708265</td>
			<td>chemiluminescence imager</td>
		</tr>
		<tr>
			<td>CO2 incubator, air jacket</td>
			<td>VWR</td>
			<td>10810-902</td>
			<td></td>
		</tr>
		<tr>
			<td>Dissecting board, 11 x 13 in</td>
			<td>Fisher Scientific</td>
			<td>09 002 12</td>
			<td></td>
		</tr>
		<tr>
			<td>Electrophoresis cell, 4-gel, for mini precast gels with mini trans-blot module</td>
			<td>Bio-Rad</td>
			<td>1658030</td>
			<td></td>
		</tr>
		<tr>
			<td>Hybridization oven</td>
			<td>Fisher Scientific</td>
			<td>UVP95003001</td>
			<td></td>
		</tr>
		<tr>
			<td>Microcentrifuge 5415 D with F45-24-11 rotor (Eppendorf)</td>
			<td>Sigma Aldrich</td>
			<td>Z604062</td>
			<td></td>
		</tr>
		<tr>
			<td>Mini dry bath</td>
			<td>Research Products International Corp</td>
			<td>400780</td>
			<td></td>
		</tr>
		<tr>
			<td>Orbital shaker</td>
			<td>VWR</td>
			<td>89032-092</td>
			<td></td>
		</tr>
		<tr>
			<td>pH meter</td>
			<td>VWR</td>
			<td>89231-662</td>
			<td></td>
		</tr>
		<tr>
			<td>Power supply for SDS-PAGE</td>
			<td>Bio-Rad</td>
			<td>1645050</td>
			<td></td>
		</tr>
		<tr>
			<td>Rectangular ice pan, maxi 9 L</td>
			<td>Fisher Scientific</td>
			<td>07-210-093</td>
			<td></td>
		</tr>
		<tr>
			<td>Stemi 508 stereo microscope with stand K LAB, LED ring light</td>
			<td>Zeiss</td>
			<td>4350649020000000</td>
			<td>dissecting microscope</td>
		</tr>
		<tr>
			<td>Timer</td>
			<td>VWR</td>
			<td>62344-641</td>
			<td></td>
		</tr>
		<tr>
			<td>Tube revolver</td>
			<td>Fisher Scientific</td>
			<td>11 676 341</td>
			<td></td>
		</tr>
		<tr>
			<td>Vortex mixer</td>
			<td>Fisher Scientific</td>
			<td>02 215 414</td>
			<td></td>
		</tr>
		<tr>
			<td>Water bath</td>
			<td>VWR</td>
			<td>89501-472</td>
			<td></td>
		</tr>
		<tr>
			<td>Western blot box</td>
			<td>Fisher Scientific</td>
			<td>NC9358182</td>
			<td></td>
		</tr>
		<tr>
			<td>Materials</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Cell culture dishes, 6 cm</td>
			<td>Fisher Scientific</td>
			<td>12-565-95</td>
			<td></td>
		</tr>
		<tr>
			<td>Cell culture plates, 12 well</td>
			<td>Fisher Scientific</td>
			<td>07-200-82</td>
			<td></td>
		</tr>
		<tr>
			<td>Cell lifters</td>
			<td>Fisher Scientific</td>
			<td>08-100-240</td>
			<td></td>
		</tr>
		<tr>
			<td>CO2</td>
			<td>Airgas</td>
			<td>CD USP50</td>
			<td></td>
		</tr>
		<tr>
			<td>Conical tubes, polypropylene, 50 mL</td>
			<td>Fisher Scientific</td>
			<td>05-539-13</td>
			<td></td>
		</tr>
		<tr>
			<td>Dumont #5 fine forceps</td>
			<td>Fine Science Tools</td>
			<td>11254-20</td>
			<td></td>
		</tr>
		<tr>
			<td>Embryo spoon</td>
			<td>Fine Science Tools</td>
			<td>10370-17</td>
			<td></td>
		</tr>
		<tr>
			<td>Microcentrifuge tubes, 0.5 mL</td>
			<td>VWR</td>
			<td>89000-010</td>
			<td></td>
		</tr>
		<tr>
			<td>Microcentrifuge tubes, 1.5 mL</td>
			<td>VWR</td>
			<td>20170-038</td>
			<td></td>
		</tr>
		<tr>
			<td>Pasteur pipet, 5.75&#34;</td>
			<td>Fisher Scientific</td>
			<td>13-678-6A</td>
			<td></td>
		</tr>
		<tr>
			<td>Pasteur pipet, 9&#34;</td>
			<td>VWR</td>
			<td>14672-380</td>
			<td></td>
		</tr>
		<tr>
			<td>Petri dishes, 10 cm</td>
			<td>Fisher Scientific</td>
			<td>08-757-100D</td>
			<td></td>
		</tr>
		<tr>
			<td>Petri dishes, 35 mm</td>
			<td>Fisher Scientific</td>
			<td>FB0875711YZ</td>
			<td></td>
		</tr>
		<tr>
			<td>Pouches, transparent, polyethylene lining</td>
			<td>Fisher Scientific</td>
			<td>01-812-25B</td>
			<td></td>
		</tr>
		<tr>
			<td>PVDF membrane</td>
			<td>Fisher Scientific</td>
			<td>IPVH00010</td>
			<td></td>
		</tr>
		<tr>
			<td>Semken forceps</td>
			<td>Fine Science Tools</td>
			<td>11008-13</td>
			<td></td>
		</tr>
		<tr>
			<td>Small latex bulb, 2 mL</td>
			<td>VWR</td>
			<td>82024-554</td>
			<td></td>
		</tr>
		<tr>
			<td>Surgical scissors</td>
			<td>Fine Science Tools</td>
			<td>14002-12</td>
			<td></td>
		</tr>
		<tr>
			<td>Syringe filter, 25 mm, 0.2 &#956;m pore size</td>
			<td>Fisher Scientific</td>
			<td>09-740-108</td>
			<td></td>
		</tr>
		<tr>
			<td>Syringe with luer tip, 10 mL</td>
			<td>VWR</td>
			<td>BD309604</td>
			<td></td>
		</tr>
		<tr>
			<td>Transfer pipet</td>
			<td>Fisher Scientific</td>
			<td>13-711-22</td>
			<td></td>
		</tr>
		<tr>
			<td>Western blot cassette opening lever</td>
			<td>Bio-Rad</td>
			<td>4560000</td>
			<td></td>
		</tr>
		<tr>
			<td>Whatmann 3MM chr chromatography paper</td>
			<td>Fisher Scientific</td>
			<td>05-714-5</td>
			<td></td>
		</tr>
		<tr>
			<td>Reagents</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>4-15% Precast protein gels, 10-well, 30 &#181;L</td>
			<td>Bio-Rad</td>
			<td>4561083</td>
			<td></td>
		</tr>
		<tr>
			<td>&#946;-glycerophosphate disodium salt hydrate</td>
			<td>Sigma Aldrich</td>
			<td>G5422-25G</td>
			<td>stock concentration 1 M</td>
		</tr>
		<tr>
			<td>&#946;-mercaptoethanol</td>
			<td>Sigma Aldrich</td>
			<td>M3148-100ML</td>
			<td></td>
		</tr>
		<tr>
			<td>Bovine serum albumin, fraction V, heat shock tested</td>
			<td>Fisher Scientific</td>
			<td>BP1600-100</td>
			<td></td>
		</tr>
		<tr>
			<td>Bromophenol blue</td>
			<td>Fisher Scientific</td>
			<td>AC403140050</td>
			<td></td>
		</tr>
		<tr>
			<td>Complete mini protease inhibitor cocktail</td>
			<td>Sigma Aldrich</td>
			<td>11836153001</td>
			<td>stock concentration 25x</td>
		</tr>
		<tr>
			<td>DC protein assay kit II</td>
			<td>Bio-Rad</td>
			<td>500-0112</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM, high glucose</td>
			<td>Gibco</td>
			<td>11965092</td>
			<td></td>
		</tr>
		<tr>
			<td>E7, mouse monoclonal beta tubulin primary antibody, concentrate 0.1 mL</td>
			<td>Developmental Studies Hybridoma Bank</td>
			<td>E7</td>
			<td>1:1,000</td>
		</tr>
		<tr>
			<td>ECL western blotting substrate</td>
			<td>Fisher Scientific</td>
			<td>PI32106</td>
			<td>low picogram range</td>
		</tr>
		<tr>
			<td>ECL western blotting substrate</td>
			<td>Genesee Scientific</td>
			<td>20-302B</td>
			<td>low femtogram range</td>
		</tr>
		<tr>
			<td>Electrophoresis buffer, 5 L</td>
			<td>Bio-Rad</td>
			<td>1610772</td>
			<td>stock concentration 10x</td>
		</tr>
		<tr>
			<td>Ethanol, 200 proof, 1 gallon</td>
			<td>Decon Laboratories, Inc.</td>
			<td>2705HC</td>
			<td>EtOH</td>
		</tr>
		<tr>
			<td>Ethylenediaminetetraacetic acid, Di Na salt dihydr. (crystalline powd./electrophor.)</td>
			<td>Fisher Scientific</td>
			<td>BP120-500</td>
			<td>EDTA</td>
		</tr>
		<tr>
			<td>Fetal bovine serum, characterized, US origin, 500 mL</td>
			<td>HyClone</td>
			<td>SH30071.03</td>
			<td></td>
		</tr>
		<tr>
			<td>Glycerol (certified ACS)</td>
			<td>Fisher Scientific</td>
			<td>G33-4</td>
			<td></td>
		</tr>
		<tr>
			<td>HRP-conjugated secondary antibody, goat anti-mouse IgG</td>
			<td>Jackson ImmunoResearch Laboratories</td>
			<td>115-035-146</td>
			<td>1:20,000</td>
		</tr>
		<tr>
			<td>HRP-conjugated secondary antibody, goat anti-rabbit IgG</td>
			<td>Jackson ImmunoResearch Laboratories</td>
			<td>111-035-003</td>
			<td>1:20,000</td>
		</tr>
		<tr>
			<td>Hydrochloric acid solution, 6N (certified)</td>
			<td>Fisher Scientific</td>
			<td>SA56-500</td>
			<td>HCl</td>
		</tr>
		<tr>
			<td>Igepal Ca - 630 non-ionic detergent</td>
			<td>Fisher Scientific</td>
			<td>ICN19859650</td>
			<td>Nonidet P-40</td>
		</tr>
		<tr>
			<td>Isopropanol (HPLC)</td>
			<td>Fisher Scientific</td>
			<td>A451-1</td>
			<td></td>
		</tr>
		<tr>
			<td>L-glutamine</td>
			<td>Gibco</td>
			<td>25030081</td>
			<td>stock concentration 200 mM</td>
		</tr>
		<tr>
			<td>Methanol</td>
			<td>Fisher Scientific</td>
			<td>A454-4</td>
			<td></td>
		</tr>
		<tr>
			<td>p44/42 MAPK (Erk1/2) primary antibody</td>
			<td>Cell Signaling Technology</td>
			<td>9102S</td>
			<td>1:1,000; anti-Erk1/2</td>
		</tr>
		<tr>
			<td>PDGF-BB recombinant ligand, rat</td>
			<td>Fisher Scientific</td>
			<td>520BB050</td>
			<td></td>
		</tr>
		<tr>
			<td>PDGF Receptor &#946; primary antibody</td>
			<td>Cell Signaling Technology</td>
			<td>3169S</td>
			<td>1:1,000</td>
		</tr>
		<tr>
			<td>Penicillin-Streptomycin</td>
			<td>Gibco</td>
			<td>15140122</td>
			<td>stock concentration 100 U/mL, 100 &#181;g/mL</td>
		</tr>
		<tr>
			<td>Phenylmethanesulfonyl fluoride, 99%</td>
			<td>Fisher Scientific</td>
			<td>AC215740100</td>
			<td>PMSF; stock concentration 100 mM</td>
		</tr>
		<tr>
			<td>Phospho-p44/42 MAPK (Erk1/2) primary antibody</td>
			<td>Cell Signaling Technology</td>
			<td>9101S</td>
			<td>1:1,000, anti-phospho-Erk1/2</td>
		</tr>
		<tr>
			<td>Phospho-PDGF Receptor &#945; /PDGF Receptor &#946; primary antibody</td>
			<td>Cell Signaling Technology</td>
			<td>3170S</td>
			<td>1:1,000</td>
		</tr>
		<tr>
			<td>Potassium chloride (white crystals)</td>
			<td>Fisher Scientific</td>
			<td>BP366-500</td>
			<td>KCl</td>
		</tr>
		<tr>
			<td>Potassium phosphate monobasic (white crystals)</td>
			<td>Fisher Scientific</td>
			<td>BP362-500</td>
			<td>KH2PO4</td>
		</tr>
		<tr>
			<td>SDS solution, 10%</td>
			<td>Bio-Rad</td>
			<td>161-0416</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium chloride (crystalline/biological,certified)</td>
			<td>Fisher Scientific</td>
			<td>S671-3</td>
			<td>NaCl</td>
		</tr>
		<tr>
			<td>Sodium fluoride (powder/certified ACS)</td>
			<td>Fisher Scientific</td>
			<td>S299-100</td>
			<td>NaF; aliquot for one time use; stock concentration 1 M</td>
		</tr>
		<tr>
			<td>Sodium orthovanadate, 99%</td>
			<td>Fisher Scientific</td>
			<td>AC205330500</td>
			<td>Na3VO4; stock concentration 100 mM</td>
		</tr>
		<tr>
			<td>Sodium phosphate dibasic anhydrous (granular or powder/certified ACS)</td>
			<td>Fisher Scientific</td>
			<td>S374-500</td>
			<td>Na2HPO4</td>
		</tr>
		<tr>
			<td>Tissue culture PBS</td>
			<td>Fisher Scientific</td>
			<td>21-031-CV</td>
			<td></td>
		</tr>
		<tr>
			<td>Transfer buffer, 5 L</td>
			<td>Bio-Rad</td>
			<td>1610771</td>
			<td>stock concentration 10x</td>
		</tr>
		<tr>
			<td>Tris base (white crystals or crystalline powder/molecular biology)</td>
			<td>Fisher Scientific</td>
			<td>BP152-1</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypsin</td>
			<td>BioWorld</td>
			<td>21560033</td>
			<td></td>
		</tr>
		<tr>
			<td>Tween 20</td>
			<td>Fisher Scientific</td>
			<td>BP337-500</td>
			<td></td>
		</tr>
		<tr>
			<td>Western blot molecular weight marker</td>
			<td>Bio-Rad</td>
			<td>1610374</td>
			<td></td>
		</tr>
		<tr>
			<td>Software</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>ImageJ software</td>
			<td>National Institutes of Health</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Animals</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Female 129S4 mice</td>
			<td></td>
			<td></td>
			<td>gift of Dr. Philippe Soriano, Icahn School of Medicine at Mount Sinai</td>
		</tr>
	</tbody>
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isolation of whole cell protein lysates from mouse facial processes and cultured palatal mesenchyme cells for phosphoprotein analysis

Mammalian craniofacial development is a complex morphological process during which multiple cell populations coordinate to generate the frontonasal skeleton. These morphological changes are initiated and sustained through diverse signaling interactions, which often include protein phosphorylation by kinases. Here, two examples of physiologically-relevant contexts in which to study phosphorylation of proteins during mammalian craniofacial development are provided: mouse facial processes, in particular E11.5 maxillary processes, and cultured mouse embryonic palatal mesenchyme cells derived from E13.5 secondary palatal shelves. To overcome the common barrier of dephosphorylation during protein isolation, adaptations and modifications to standard laboratory methods that allow for isolation of phosphoproteins are discussed. Additionally, best practices are provided for proper analysis and quantification of phosphoproteins following western blotting of whole cell protein lysates. These techniques, particularly in combination with pharmacological inhibitors and/or murine genetic models, can be used to gain greater insight into the dynamics and roles of various phosphoproteins active during craniofacial development.

When attempting to characterize the phosphorylation of proteins isolated from mouse facial processes and/or cultured palatal mesenchyme cells, the representative results will ideally reveal a distinct, reproducible band following western blotting with an anti-phosphoprotein antibody that runs at or near the height of the corresponding total protein band (Figure 3). However, if extensive phosphorylation of the protein occurs, there may be a slight upward shift of the phosphoprotein band compa...

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Isolation of Whole Cell Protein Lysates from Mouse Facial Processes and Cultured Palatal Mesenchyme Cells for Phosphoprotein Analysis

The protocol presents a method for isolating whole cell protein lysates from dissected mouse embryo facial processes or cultured mouse embryonic palatal mesenchyme cells and performing subsequent western blotting to assess phosphorylated protein levels.

Mammalian craniofacial development is a complex morphological process during which multiple cell populations coordinate to generate the frontonasal ...

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