Anti fakpy397

The following antibodies were used: anti-Src, anti-pSrcY416, anti-FAKpY397, anti-pPaxillin Y118, anti-Paxillin, anti-pCortactin Y486, anti-pCortactin Y466, anti-Cortactin were all from life technologies; Anti-GRP78 N20, antiGRP78 C20 (for antibody blocking), anti-β-actin were from Santa Cruz. Anti-GRP78 (for immunoprecipitation and in cell western analysis), anti-pEGFR Y1101, anti-pEGFR Y1068, anti-pEGFR Y845, anti-EGFR, anti-p-Tyr and rabbit isotype IgG were obtained from Abcam. Anti-EGFP was obtained from Origen. All the secondary antibodies except for IRDYE680RD-conjugated antibody (LI-COR) were all from abcam. α2M was purchased from Sigma-Aldrich and activated as previously reported [18 (link)]. PP2, lipofectamine 2000, TRITC-WGA and fibronectin were from life technologies. Protein A/G agarose beads and G-sepharose beads were purchased from GE healthcare. RT-PCR kit was from Takara. Plasma protein isolation kit was purchased from Pierce. GRP78-EGFP recombinant and corresponding pEGFP-N1 were kindly given by the Cell Biology Department of China Medical University.

Cells were harvested into RIPA buffer (50 mM Tris HCl pH 8.0; 150 mM NaCl; 1% NP-40 0.5% sodium deoxycholate; 0.1% SDS) supplemented with protease/phosphatase inhibitor cocktail (Roche). Twenty micrograms of whole cell lysate were separated by electrophoresis in 10% Bis-Tris polyacrylamide gel followed by blotting to nitrocellulose membrane. Non-specific binding sites were blocked by incubating the blots for 2 hrs at room temperature with 5% (w/v) non-fat dry milk in PBS. Blots were incubated overnight with primary antibodies at the following concentrations: anti-ASPN (1:1000; # HPA008435; Sigma-Aldrich, Protein Atlas; plus other antibodies specified in Supplementary Table S2), anti-FAK pY397 (1:500, Life Technologies), and anti-GAPDH (1:25000; Sigma-Aldrich) was used as a loading control. Secondary antibodies were as follows anti-rabbit IgG, HRP-linked Antibody (#7074) and Anti-mouse IgG, HRP-linked Antibody (#7076), both purchased from Cell Signaling Technology, MA, USA. Signal detection was performed with Dura/ Femto ECL western blotting substrate (ThermoFisher Scientific). Analysis of optical density was performed using ImageJ software.