Anti pigf1r β

Once whole cell or nuclear and non-nuclear protein fractions were obtained, the protein concentration in the supernatants was determined using Bradford reagent. Fifty micrograms of proteins was resolved by SDS-PAGE and transferred to polyvinylidene fluoride membranes. Blots were blocked and probed with various antibodies: anti-IGF1R β (#3027), anti-pIGF1R β (#3918), anti-AKT (#9272), anti-pAKT (#9271), anti-MAPK 42-44 (#9102), anti-pMAPK 42-44 (#9101), anti-S6K (#2708), anti-pS6K (# 9205), and anti-β actin (#4970) from Cell Signaling Technology (Boston, MA, USA); anti-lamin B (SC-6217) from Santa Cruz Inc. (Dallas, TX, USA); anti-pPDC subunit E1-α (NB110-93479) and ACSL5 (H00051703-M01) from Novus Biologicals (Centennial, CO, USA); anti-FAS (610962) from BD Biosciences (San Jose, CA, USA); and anti-GAPDH (MAB374) from Millipore (Darmstadt, Germany).

Tissues and cells were homogenized. Solubilized proteins in lysis buffer (0.1 M Tris–HCl (pH 6.8), 4% SDS, 20% glycerol, 12% 2‐mercaptoethanol, and BPB) were subjected to SDS–PAGE, and proteins were electrotransfered to nitrocellulose membranes. Immunodetection was carried out using an ECL kit (GE Healthcare) according to the manufacturer's protocol. Antibodies used were as follows: anti‐β‐catenin (1:5,000, BD, #610154), anti‐P‐β‐catenin (1:2,000, Cell Signaling Technology, #4176), anti‐active‐β‐catenin (1:2,000, Cell Signaling Technology, #19807), anti‐PTEN (1:2,000, Cell Signaling Technology, #9559), anti‐P‐PTEN (1:2,000, Cell Signaling Technology, #9554), anti‐AKT (1:1,000, Cell Signaling Technology, #9272), anti‐P‐AKT (1:1,000, Cell Signaling Technology, #9271), anti‐IGF‐1Rβ (1:1,000, Cell Signaling Technology, #3018), anti‐P‐IGF‐1Rβ (1:1,000, Cell Signaling Technology, #4568), and anti‐HSC70 (1:2,000, Santa Cruz Biotechnology, #sc7298).

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.