Socs1

Nuclear/cytoplasmic fractionation was separated using the Cell Fractionation Kit (Cell Signaling Technology, USA) according to the manufacturer’s instructions, and the whole cell lysates were extracted with RIPA Buffer (Cell Signaling Technology). Western blotting was performed according to a standard method, as described previously [35 (link)]. Antibodies against E-cadherin (Cat# 3195), Vimentin (Cat# 5741), Fibronectin (Cat# 4706), SOCS1 (Cat# 3950), TNIP1 (Cat# 4664) and PIAS4 (Cat# 4392) were purchased from Cell Signaling Technology, and p65 (cat# 10745–1-AP) from Proteintech, p84 (Cat#:PA5–27816) from Invitrogen and PDLIM7 (Cat#:SAB1406807) from Sigma-Aldrich,USA. The membranes were stripped and reprobed with an anti–α-tubulin antibody (Sigma-Aldrich, USA) as the loading control.

Nuclear/cytoplasmic fractionation was separated by using Cell Fractionation Kit (Cell Signaling Technology, USA) according to the manufacturer's instructions, and the whole cell lysates were extracted using RIPA Buffer (Cell Signaling Technology). Western blot was performed according to a standard method, as described previously [60 (link)]. Proteins were visualised using ECL reagents (Pierce, USA). Antibodies against Bcl-2 (Cat#: 2872), Bcl-xL (Cat#: 2764), pSTAT3 (Cat#: 9145), SOCS1 (Cat#: 3950), SOCS2 (Cat#: 2779), SOCS3 (Cat#: 2923) were purchased from Cell Signaling Technology and p84 (Cat#: PA5-27816), SOCS4 (Cat#: PA5-21599) and SOCS5 (Cat#: PA5-21600) from Invitrogen. The membranes were stripped and reprobed with an anti–α-tubulin antibody (Cell Signaling Technology. Cat#: 2125) as the loading control.

This procedure was performed as described previously14 (link). Each membrane was incubated overnight with the respective primary antibody [IRS-1 and Phosphatidylinositol-4,5-bisphosphate 3-kinase p85 alpha subunit (PI3Kp85α) (Upstate Biotechnology, Millipore, Lake Placid, USA), Phosphatidylinositol-4,5-bisphosphate 3-kinase p110 beta subunit (PI3Kp110β), Protein kinase B1 (AKT1), Protein kinase B2 (AKT2), Suppressor of cytokine signaling protein 1 (SOCS1) and Suppressor of cytokine signaling protein 3 (SOCS3) (Cell Signaling, UK) and Insulin receptor beta (IRβ), protein kinase C-ζ (PKCζ) and Glucose transporter type 4 (GLUT4) receptor (Santa Cruz Biotechnology, Germany)]. Following washing with TBS/T solution (1 X TBS, 0.05% Tween 20), membranes were incubated with horseradish peroxidase-conjugated anti-rabbit or anti-mouse antibody as appropriate (Jackson Immuno Research, UK). Antibody binding was detected using Super Signal West Pico Chemiluminescent substrate (Thermo Scientific, UK) with an ImageQuant^TM LAS 4000 machine and quantified using ImageQuant^TM LAS 4000 software (GE Healthcare, UK). Coomassie Blue staining was used to confirm equal loading and GAPDH (Santa Cruz Biotechnology, Germany) antibody blotted on each membrane to further confirm equal loading.

The homogenization buffer contained 20 mM MOPS, 50 mM β-glycerophosphate, 5 mM EDTA, 1 mM DTT, 1 mM sodium vanadate, 1 mM PMSF, and 50 mM NaF. Tissue was mixed in the homogenization buffer and sonicated on the ice. The protein concentration in the supernatent isolated by centrifuging was determined by the Bradford assay (Thermo). Proteins were separated with SDS-PAGE and then transferred to PVDF membranes on the ice (Bio-Rad). The membranes was incubated with appropriate primary antibody overnight at 4 °C. After three times washed, the appropriate secondary antibody was added and incubated for 1 h at room temperature. After three times further washing, the enhanced chemiluminescence detection buffer (Amersham) was dripped on the membrane. Signal was detected by Chemiluminescence Apparatus (Bio-Rad, CA). GAPDH was used as reference. All experiments were repeated at least three times. The following antibodies were used: SOCS-1, STAT1 and p-STAT1 (Cell Signaling Technology, Beverly, MA).

Total muscle protein was extracted using RIPA buffer containing protease inhibitor cocktail (Roche, Indianapolis, IN, USA) and 1 mM PMSF. Protein concentrations were measured by a DC protein assay (Bio-Rad). Western blotting was performed by standard protocol. The following antibodies were used: SOCS1 (1 : 1000, Cell signaling, Danvers, MA, USA), phospho-STAT3 (1 : 1000, Cell signaling), anti-mouse STAT3 (1 : 1000, Cell signaling), γ-tubulin (1 : 5000, Sigma-Aldrich), GAPDH (1 : 5000, Sigma-Aldrich). Primary antibody was visualized with either IRDye 680RD goat anti-mouse or IRDye 800CW goat anti-rabbit (LI-COR, Lincoln, NE, USA) on the Odyssey imaging system (LI-COR Biosciences).