Ent. SW480 was established from the primary tumor, SW620 from a
Ent. SW480 was established from the primary tumor, SW620 from a lymph node metastasis. A ?D: 20 g of TCL from each cell line were separated by PubMed ID:https://www.ncbi.nlm.nih.gov/pubmed/25957400 SDS-PAGE and western blots (WB) conducted with antibodies as indicated. Arrowheads in A indicate pY bands increased or only detectable in SW620 cells. The asterisk in C indicates a barely visible band that comigrates with the expected size of Lck. Actin levels were analysed as loading control in D. To determine the phosphorylation of Lck in the activation loop (results shown in E), which critically regulates Lck activity, 1 mg of TCL from each cell line was immunoprecipitated with anti-Lck mAb and probed with anti-pY419Src that cross-reacts with several SFK family members, including Lck, due to high conservation of this epitope (further details in [5]). An IgG control of the same isotype was also included to precipitate non-specific binding proteins from 1 mg of TCL of SW620 cells. Note that some of the molecular weight markers used do not run exactly accordingly to the indicated molecular weights of the marker proteins, presumably due to the coupling of dye molecules to them.SW31 -EIP: Lck(page number not for citation Procyanidin B1 supplier purposes)SWPage 4 ofCell Communication and Signaling 2008, 6:http://www.biosignaling.com/content/6/1/PLCgSH2(N)Shp2SH2(C)PI3KSH2(C)220 -97 -66 -45 -30 -21 WB: pY (4G10) 45 -30 CB-stainFigure 3 Differential binding of pY proteins from SW620 cells to SH2 domains from 15 different signalling proteins Differential binding of pY proteins from SW620 cells to SH2 domains from 15 different signalling proteins. Top panel: 1 mg of TCL was precipitated with 50 g of bead-immobilised GST-SH2 fusion protein, precipitates repeatedly washed with RIPA buffer and bound proteins analysed by anti-pY western blotting. Bottom panel: Coomassie blue staining of the affinity purified GST-SH2 domain fusion proteins used for the precipitations. Approximately 10 g of each protein was loaded onto the gel to analyse protein purity and integrity.SW620 TCLPage 5 ofGapSH2(N)TensinSHMonaSH(page number not for citation purposes)Vav1SHGrapSHCrkLSHLck-SHNckSHShcSHFynSHSrcSHAblSHGSTCell Communication and Signaling 2008, 6:http://www.biosignaling.com/content/6/1/LckSH2, which had been pre-incubated with an excess of a phosphopeptide (EPQpYEEIPI; originally derived from hamster middle T antigen) that binds with high affinity to the pY binding pocket of the LckSH2 domain [8,9], or GSH beads alone. Bound proteins were detected with Coomassie Blue staining. A representative example is depicted in Figure 4. These experiments showed that most of the SW620 S100 proteins that interact with the LckSH2 require indeed the pY binding pocket of this domain. The gel lanes containing the proteins precipitated with GSTLckSH2 in the absence of pY peptide were subsequently cut into 10 slices, which were subjected to tryptic digest and MS analyses. The proteins identified through this approach are listed in Additional file 2.LckSH2 affinity chromatography allows rapid mass spectrometric identification of numerous tyrosine kinase substrates Notably, many of the proteins identified are readily known as substrates of protein tyrosine kinases. Previously detected phosphosites for the MS-identified proteins were extracted from the PhosphoSitePlusTM database http://www.phosphosite.org and are also listed in Additional file 2. As this database is constantly updated and several other phosphoprotein databases exist, this listing is expected to und.