Igf 1 receptor β

For western blot analysis, differentially treated cells were washed with cold PBS and then lysed in lysis buffer containing protease and phosphatase inhibitor. Insoluble cell fragments were removed by centrifugation. Cells were separated by SDS-PAGE electrophoresis and next transferred to PVDF membranes. Membranes were incubated overnight with the primary antibodies against phospho-AKT (Cell Signaling Technologies), ERK-1/2 (Cell Signaling Technologies), phosphor-ERK-1/2 (Cell Signaling Technologies), STAT3 (Cell Signaling Technologies), phospho-STAT3 (Cell Signaling Technologies), phospho-IGF-I (Cell Signaling Technologies, Danvers, MA, USA), JAK1, phospho-Jak1 (Tyr1022/1023) (Cell Signaling Technologies), JAK2 (Cell Signaling Technologies), phospho-Jak2 (Tyr1007/1008) (Cell Signaling Technologies), IGF-I Receptor β (Cell Signaling Technologies), AKT (Cell Signaling Technologies), epithelial-mesenchymal transition (EMT) antibody sampler kit (Cell Signaling Technologies). Fluorescent secondary anti-mouse (Thermo Scientific, Waltham, USA) and anti-rabbit (Thermo Scientific, Waltham, USA) antibodies were used and were detected using Odyssey Sa infrared imager (LI-COR Biosciences, Lincoln, USA).

Following treatment with drugs, cells were lysed and whole cell fractions were isolated as previously described [17 (link)]. Proteins (10–30 μg) were separated by 10%–12% (w/v) polyacrylamide gel electrophoresis and electroblotted to PVDF membranes. After blocking with non-fat milk for 1 h, membranes were incubated overnight with the following primary antibodies (1:1000 dilution) from Cell Signaling Technology (Danvers, MA, USA): E-Cadherin (Cat #3195), N-Cadherin (Cat #13116), TCF8/ZEB1 (Cat #3396), β-Catenin (Cat #8480), Vimentin (Cat #5741), phospho-Akt (Ser473) (Cat #4060), Akt (Cat #9272), phospho-IGF-I Receptor β (Tyr1135/1136) (Cat #3024), IGF-I Receptor β (Cat #9750), phospho-PI3 Kinase p85 (Tyr458)/p55 (Tyr199) (Cat 4228), PI3 Kinase p85 (Cat 4257), phospho-mTOR (Ser2448) (Cat #5536), mTOR (Cat #2983). β-Actin (Cat #8457) was used at the same time as a loading control. After incubation with the secondary antibody (HRP-conjugated; 1:5000 dilution) for 1 h, at room temperature, the conjugates were developed and visualized using a Molecular Imager FX^TM System (BioRad; Hercules, CA, USA).