Anti yes

The following mouse monoclonal antibodies were used: anti-phosphotyrosine PY99 (Santa Cruz Biotechnology), anti-phosphotyrosine (4G10), anti-α9β1 antibody (Y9A2), anti-integrin α4 (HP2/1), anti-total β1 (MAB1959), anti-active β1 (12G10), anti-myc tag (4A6), anti-v-Src (OP07), anti-PTEN (6H2.1), anti-pan-actin (all from Millipore), activating β1-integrin antibody (TS2/16, Thermo Scientific), anti-Yes, anti-Fyn, anti-Hck, anti-Crk (all from BD Biosciences), anti-cellular/EDA fibronectin antibody (FN-3E2, Sigma). The following rabbit polyclonal antibodies were used: anti-cortactin (ab11066 from Abcam, for G361 lysates) and anti-cortactin phospho-Y470 (ab51703, from Abcam, or sc-101661 from Santa Cruz), anti-cortactin phospho-Y421 and anti-Src family kinases (SFK) phospho-Y416 (both from Cell Signaling), anti-arg, anti-Pyk2, anti-FAK (all from Millipore), anti-FAK phospho Y397 (Invitrogen), and anti-fibronectin (R2/7, made against bovine plasma fibronectin53 (link)). Secondary antibodies used were fluorochrome-conjugated antibodies (Alexa Fluor 488 and 546, Invitrogen) and horseradish peroxidase (HRP)-conjugated antibodies (Dako).

The protein concentration of each fraction was measured by a Bradford protein assay. An amount of 5 µg protein of each fraction was separated on 10% polyacrylamide gels at 40 mA per gel for 2 h and were blotted onto nitrocellulose membranes by semi-dry Western blot at 180 mA per membrane for 1 h. Membranes were blocked in 5% skim milk powder in TBS-T, incubated with the indicated primary antibody solution (in TBS-T supplemented with 5% BSA and 0.1% sodium azide) at 4 °C overnight followed by incubation with corresponding HRP-conjugated secondary antibody solution (in 3% BSA in TBS-T) at RT for 1 h. Proteins were visualized with ECL Detection Reagents (GE Healthcare, Chicago, IL, USA). The antibodies used were anti-beta Actin (MP, Irvine, CA, USA), anti-Tapasin, anti-beta2-Microglobulin, anti-ERp57, anti-Calreticulin (all Abcam, Cambridge, UK), anti-Yes (BD Bioscience), anti-Flotillin1, anti-Caveolin1 (Sigma-Aldrich, Seelze, Germany), anti-TAP1 (Novus Biologicals, Cambridge, UK), anti-TAP2 (MBL, Woburn, MA, USA), HRP-conjugated goat anti mouse and HRP-conjugated goat anti-rabbit (Thermo Scientific, Bonn, Germany).

The following antibodies were used: anti-Src (05–184; Merck, Darmstadt, Germany), anti-Yes (610375; BD Biosciences, San Jose, CA, USA), anti-Lyn (sc-7274; Santa Cruz Biotechnology, Dallas, TX, USA), anti-Csk (610080; BD Biosciences), anti-glyceraldehyde 3-phosphate dehydrogenase (GAPDH) (2275-PC-100; R&D Systems, Minneapolis, MN, USA), anti-p-Tyr (05–1050; Merck), anti-Src family phospho-Y418 (p-SFKs A-loop; ab40660; Abcam, Cambridge, UK), anti-p-Src Y530 (sc-166860; Santa Cruz Biotechnology), anti-p-Lyn Y508 (CSB-PA000691; Cusabio, Wuhan, China)), anti-Fyn (sc-434; Santa Cruz Biotechnology), anti-E-cadherin (#3195; Cell Signaling Technology, Danvers, MA, USA), anti-vimentin (V6630; Sigma-Aldrich), anti-Cbl-b (sc-8006; Santa Cruz Biotechnology) and anti-c-Cbl (sc-1651; Santa Cruz Biotechnology). Horseradish peroxidase-conjugated anti-mouse IgG (#7076; Cell Signaling Technology) and anti-rabbit IgG (711-035-152; Jackson Immunoresearch, West Grove, PA, USA) antibodies were used for Western blotting.