Anti igf1rβ

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).

Recombinant CYR61 and SP600125 were purchased from Sigma-Aldrich (Lyon, France), recombinant IGF-1 was purchased from R&D Systems (Lille, France).
Neutralizing anti-IGF1 antibody was purchased from R&D Systems. Rabbit polyclonal anti-CYR61 and anti-pIGF1Rβ were purchased from Abcam (Cambridge, UK), anti-Actin was purchased from Sigma Aldrich and anti-IGF1Rβ, anti-pGSK3β, anti-pJNK, anti-JNK were purchased from Cell Signaling Technology (Saint Quentin en Yvelines, France). Mouse monoclonal anti-E-cadherin was purchased from Santa Cruz Biotechnologies (Santa Cruz, CA, USA), anti- GSK3β was purchased from Cell Signaling Technology, and anti-IGF1 was purchased from R&D Systems.

Anti-NDRG1 antibody was generated as previously described [43 (link)]. Other antibodies were purchased as follows: anti-α-tubulin antibody was from Sigma-aldrich Co; anti-EGFR, anti-phospho-EGFR (Tyr1068), anti-phospho-HER3, anti-Met, anti-phospho-Met, anti-PDGFRβ, anti-phospho-PDGFRβ, anti- IGF-1Rβ, anti-phospho-IGF-1Rβ, anti-ERK1/2, anti-phospho-ERK1/2, anti-AKT, anti-phospho-AKT (Thr308 and Ser473), anti-mTOR, anti-phospho-mTOR (Ser2448 and Ser2481), anti-Raptor, anti-Rictor, anti-S6K, anti-phospho-S6K(Thr389), anti-phospho-S6, anti-PTEN, anti-phospho-PTEN, anti-IRS-1, anti-PDK1, anti-PARP, anti-phospho-PDK1, anti-TSC-1, anti-TSC-2, and anti-phospho-4EBP-1 antibodies were from Cell Signaling Technology (Beverly, MA); anti-HER2 and anti-HER3 antibodies were from Santa Cruz Biotechnology (Santa Cruz, CA); anti-glyceraldehyde-3-phosphate dehydrogenase (GAPDH) antibody was from Trevigen Inc. (Gaithersburg, MD): anti-p27 antibody was from BD Biosciences (San Jose, CA): anti-cleaved PARP antibody was from Promega (Madison, WI): anti-phospho-HER2 antibody was from Millipore (Billerica, MA): anti-β-actin was from Abcam (Cambridge, UK).

Western blot analysis was performed as described previously [5 (link),6 (link)] using anti-occludin (NBP1-87402) obtained from Novusbio, Total OXPHOS Rodent WB Antibody Cocktail (ab110413) and anti-PGC-1α (ab54481) obtained from Abcam (Cambridge, UK), anti-HSP60 (sc-376240) obtained from Santa Cruz and anti-phospho-SAPK/JNK (Thr183/Tyr185) (#9251), anti-JNK2 (#9258), anti-SOD2 XP (#13141), anti-SIRT3 (#5490), anti-phospho-AKT (Ser473) (#9271), anti-AKT (#9272), anti-IRβ (#3025) and anti-IGF-1Rβ (#3027) antibodies, as well as the secondary antibodies anti-rabbit antibody (#7074) and anti-mouse antibody (#7076) obtained from Cell Signaling (Cambridge, UK). Ponceau staining served as a loading control. Oxyblot analysis was carried out as previously published [33 (link)] with anti-DNP antibody after membrane derivatization (D9656, Sigma-Aldrich, Taufkirchen, Germany). Specific bands were detected by using a chemiluminescence reagent in the ChemiDoc™ Imaging System with ImageLab software (Bio-Rad, Munich, Germany). Band intensities were quantified via densitometric analysis using Image Lab 5.2.1 and Image J software and were normalized to protein content exemplified by Ponceau staining or total unphosphorylated proteins (JNK and AKT phosphorylation).