P shc

The antibodies used for Western blotting included phosphorylated (p)-Smad2 (S465/467, Cell Signaling Technology), Smad2 (Cell Signaling Technology), p-Shc (Y239,240, Cell Signaling Technology), Shc (Abcam), PlxnD1 (Thermo Fisher Scientific), PECAM-1 (Santa Cruz Biotechnology), Alk5 (Sigma-Aldrich for biochemical assays and R&D for tensional force application), TGFβ1 to TGFβ3 neutralization antibody (R&D), Lamin B1 (Abcam), and heat shock protein 90 (Hsp90; Cell Signaling Technology). The inhibitor SB431542 (Sigma-Aldrich) was used at a concentration of 10 μM. Recombinant mouse TGFβ2 was purchased from R&D Systems (Bio-Techne) and used at 5 ng/ml.

Protein aliquots of 25 μg each were resolved by SDS polyacrylamide gel electrophoresis (Bio-Rad, Hercules, CA) or 1000 μg aliquots of total proteins were immunoprecipitated with the appropriate antibodies. The immune complexes were recovered with Protein G-Sepharose or Protein A-Sepharose beads (Zymed Laboratories, California). Electrophoresed protein samples or immunoprecipitated samples were transferred to polyvinylidene difluoride membranes (Bio-Rad). After washing three times, the membranes were incubated with blotting-grade blocker (Bio-Rad) for 1 h at room temperature and overnight at 4 °C with primary antibodies to p-AXL (Tyr702), t-AXL, p-EGFR, p-IGF-1R, t-IGF-1R, p-Akt (Ser473), t-Akt, p-Gab1, t-Gab-1, p-Shc, t-Shc, IRS-1,β-actin (13E5) (1:1,000 dilution; Cell Signaling Technology, Danvers, MA, USA), p-Erk1/2 (Thr202/Tyr204), t-Erk1/2, and t-EGFR (1:1000 dilution, R&D systems).
After washing three times, the membranes were incubated for 1 h at room temperature with HRP-conjugated species-specific secondary antibody. Immunoreactive bands were visualized using SuperSignal West Dura Extended Duration Substrate Enhanced Chemiluminescent Substrate (Pierce Biotechnology). Each experiment was independently performed at least three times.