Fak antibody

Total β-catenin antibody (1:1000) was purchased from Invitrogen (712700), glyceraldehyde-3-phosphate dehydrogenase antibody (1:1000) and FAK antibody (1:1000, 3285) were obtained from Cell Signaling Technologies, anti-ATP6V0a3 antibody (1:500) was obtained from Novus (nbp1-89333, 1:1000). CD63 antibody was obtained from Abcam.
Antibodies against Pak1 (ab131522), Ras (ab52939), GSK3 (ab93926, 1:4000), and secondary antibodies for immunostaining for cells (ab150120, ab150084, ab150117, ab150081) (1:300) were obtained from Abcam. Antibody against the FA protein Tes (HPA018123, 1:100) was obtained from Atlas antibodies. Secondary antibodies for immunostaining arrays (A-11001, A-11011 were obtained from Invitrogen). HRP-linked secondary antibodies 7076, 7074 at 1:5000 (Cell Signaling) were used for western blots and analyzed with an iBright Imaging system. EIPA (A3085), and LiCl (L4408), were obtained from Sigma. Baf (S1413) and PF-00562271 (FAK inhibitor, S2672) were purchased from Selleckchem. TMR-dextran 70 kDa was obtained from Thermo Fisher (D1818). PMA (1201) was purchased from TOCRIS.

Total protein was obtained using RIPA buffer with cocktail inhibitors (Cell Signaling Tech, USA). Protein concentration was measured using a BCA kit (Pierce, USA). Equal amounts of protein were separated on a 15% gel and then transferred to 0.22 μm PVDF membranes (AmerSham, USA). The membranes were blocked in 5% bovine serum albumin (BSA) in Tris-buffered saline with Tween 20 buffer (TBST) for 2 h and then incubated overnight at 4 °C with the following primary antibodies: FAK-antibody (1:1000, Cell Signaling Tech, USA) and anti-β-actin (1:1000, Cell Signaling Tech, USA). Then, the membranes were washed three times with TBST and incubated with horseradish peroxidase-conjugated secondary antibody (goat anti-rabbit IgG, 1/5000 or goat anti-mouse IgG, 1:5000, Cell Signaling Tech, USA) for 1 h at room temperature. Blots were developed using a chemiluminescence kit (Pierce, USA) and exposed to X-ray film. The bands on the film were scanned and analyzed with Quality One software (Bio-Rad). Primary antibodies and conditions used to probe blots were rabbit anti-FAK (1:1000; Cell Signaling, USA), mouse anti-β-actin (1:1000; Cell Signaling, USA). Appropriate HRP-conjugated secondary anti-rabbit or anti-mouse antibodies were used (Cell Signaling, USA). The FAK protein expression level was normalized to β-actin expression on the same nitrocellulose membrane.