P pdgfra

GIST-T1 cells were treated with CM from CAFscr, CAFshPDGFC #1, or CAFshPDGFC #2 for 24 h. The lysates with RIPA buffer (Cell Signaling Technology, Beverly, MA) were subjected to Western blot with the antibodies (1:1000 dilutions) against p-PDGFRA (Cell Signaling Technology, 4547P), PDGFRA (Cell Signaling Technology, 3164S), p-PDGFRB (ABclonal, Woburn, MA, Apo815), PDGFRB (Cell Signaling Technology, 3169 S), and β-actin (Cell Signaling Technology, 4967S). After SDS–polyacrylamide gel electrophoresis (Invitrogen) with the lysates, the gel was transferred to polyvinylidene difluoride membrane (Bio-Rad) with Trans-Blot Turbo transfer system 690BR (Bio-Rad). The immune-reactive bands with the secondary HRP-conjugated antibodies were visualized using a chemiluminescent substrate (Invitrogen) and were exposed to X-ray film (Genesee Scientific, San Diego, CA).

For Western Blots (WB), NSCs were extracted in RIPA lysis buffer (NEB) supplemented with protease inhibitors (Sigma). For immunoprecipitation (IP) cells were lysed in IP buffer (20mM sodium phosphate buffer, 1 mM EDTA, 0.2% NP40, 150 mM NaCl supplemented with Na-orthovanadate, PMSF, NaF and protease inhibitor mixture [Sigma]). After sonication and centrifugation, supernatant was incubated overnight at 4°C with Pdgfra antibody, followed by 2-hr incubation with Dynabeads M-280 (Invitrogen), washed with IP buffer (0 mM NaCl, 150 mM NaCl and 1 M NaCl) and eluted in Laemmli sample buffer. All primary antibodies were used at 1:1000 dilution and secondary antibodies at 1:10000. The following antibodies were used: p53, Pdgfra, p-Pdgfra, p-Chk2, p-Akt, Akt (all Cell Signaling); Myc-9E10 (CRUK); Smc1 (Abcam); p-Smc1, Chk2, Tubulin (all Merck); p-Kap1, Kap1 (both Bethyl Labs); Atm (Santa Cruz), p-Atm (Epitomics), p-p53, Actin, GAPDH, HRP-conjugated goat anti-mouse/rabbit IgG (all Sigma).