Anti p pdgfrβ

CBA was obtained from Sigma (St. Louis, MO, USA). The structure of CBA is illustrated in Fig. 1a. The 3-(4,5-Dimethylthiazol‑2‑yl)-2,5-diphenyltetrazolium bromide (MTT) was from Sigma (St. Louis, MO, USA). PDGF‑BB was obtained from PeproTech (Seoul, Korea). Gelatin was obtained from Difco (Lexington, KY, USA). Lipofectamine 2000 reagent was purchased from Invitrogen. Anti-p-PDGFRβ (Cat. no. 4549) and anti-p‑Akt (Ser473; Cat. no. 4058) antibodies were obtained from Cell Signaling Technology, Inc. (Beverly, MA, USA). Anti-p-ERK (Cat. no. sc‑7383), anti-ERK (Cat. no. sc‑93), anti-Akt (Cat. no. sc‑5298) and anti-α-tubulin (Cat. no. sc‑5286) antibodies were from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA). All the chemicals not mentioned above were obtained from Sigma.

Cultured cells were lysed with cell lysis buffer (Beyotime). Protein samples were separated by precast NuPAGE Novex 4–12% (w/v) Bis–Tris gels (Life Technologies) and then transferred on to nitrocellulose membrane using the iBlot™ dry blotting system as described by the manufacturer (Life Technologies). Membranes were stained with Ponceau S and blocked in TBST buffer (20 mM Tris, pH 7.5, 150 mM NaCl, 0.1% tween 20) containing 5% non-fat dry milk for 1 h at room temperature. The blots were reacted with primary antibodies (anti-PCNA, anti-p-p27, anti-p27, anti-p21, anti-p-ERK, anti-ERK, anti-p-p38MAPK, anti-p38MAPK, anti-p-JNK, anti-JNK, anti-p-PDGFRβ, anti-PDGFRβ, anti-cyclin D1, anti-cyclin E, anti-CDK2, 6, Cell Signaling Technology; anti-CDK4 and anti-GAPDH, abcam) overnight at 4°C, then with a secondary antibody conjugated with HRP (Cell Signaling Technology) for 2 h at room temperature. Blots were developed using chemiluminescence (ECL, Thermo Fisher Scientific) on Fluor Chem M image system (Protein simple).

Erlotinib was kindly provided by F. Hoffman-La Roche Ltd, gefitinib was provided by AstraZeneca Inc; VS-4718 was provided by Verastem Inc; afatinib, osimertinib, lapatinib, AZD4547, and BIBF1120 were purchased from Selleck Chemicals; SU11274 and picropodophyllin were from Carbiochem; dasatinib was from Bio Vision; SB203580 was from Cayman Chemical; sorafenib was acquired from Toronto Research Chemicals Inc, cisplatin was from Bristol-Myers Squibb Company; and PD173074 was from Sigma-Aldrich.
Anti-HER2 and anti-pHER2 antibodies were purchased from Merck Millipore Corporation, anti-EGFR, anti-pEGFR, anti-pHER3, anti-HER4, anti-pHER4, anti-pc-Met, anti-IGF1Rβ, anti-pIGF1Rβ, anti-PDGFRβ, anti-pPDGFRβ, anti-FGFR1, anti-pFGFR, anti-ERK1/2, anti-pERK1/2, anti-AKT, anti-pAKT, anti-STAT3, anti-pSTAT3, anti-PTEN, anti-SRC, anti-FYN, anti-LYN, anti-YES, anti-LCK, anti-pSRC family (Y416), anti-pSRC (Y527), anti-FAK, anti-pFAK (Y397), anti-pFAK (Y576/577), anti-pFAK (Y925) and anti- EGFR (del E746-A750) antibody were from Cell Signaling Technology, anti-HER3 and anti-c-Met were from Santa Cruz Biotechnology Inc, anti-β-actin was from Abcam, Inc., and anti-α-tubulin was from Sigma-Aldrich.