Anti irs2

Male mice (6–8 weeks old; n = 6–7/group) were randomised to a standard diet (16.7% kJ fat and 12.4% kJ sugar wt/wt) or high-fat/high-sugar diet (HFHS; 49.2% kJ fat and 32.2% kJ sugar wt/wt) for 15 weeks. Body weight and food consumption were measured weekly. At the end of the study, GTTs and metabolic measurements were performed. Metabolic measurements were measured using a Columbus Instruments Comprehensive Lab Monitoring System (CLAMS; Columbus Instruments), as described in ESM. Tissues (pancreas, liver and white adipose tissue) were dissected, weighed and flash frozen or fixed for immunohistochemistry (anti-CD3, anti-CD68, anti-thioredoxin-interacting protein [TXNIP] and anti-phospho [p][S536]-p65 [1:100; Abcam], anti-ceramide [1:10; Enzo Life Sciences]). Immunoprecipitated insulin receptor (IR) or IRS-2 from liver protein extracts (anti-IRS2 [Cell Signaling], anti-IR [Santa Cruz Biotechnology]; 1 μg) were subjected to Tris-glycine PAGE, immunoblotted with anti-IRS2 or anti-p-Tyr (1:1000; Santa Cruz Biotechnology), visualised by enhanced chemiluminescence and quantified using ImageJ.

The following commercially available primary antibodies were used for immunoblotting: anti-IRS2 (Cell Signaling Technologies, 4502) 1:750; anti-Cdh1/Fzr1 (Sigma Aldrich, CC43) 1:500; anti-anillin (a gift from Christine Field (21 (link))) 1:1000; anti-Aurora B (Bethyl, A300-431) 1:1000; anti-Eg5 (Cell Signaling Technologies, 4203) 1:1000; anti-Top2A (Cell Signaling Technologies, 12286) 1:1000; anti-TK1 (Cell Signaling Technologies, 8960) 1:1000; anti-Mps1 (Abcam, ab11108), 1:1000); anti-APC3 (BD Transduction Laboratories, 610455) 1:500; anti-cyclin B1 (Santa Cruz Biotechnology, sc-752) 1:500; anti-Cdc20 (Santa Cruz Biotechnology, sc-8358) 1:500; anti-c-Myc (9E10, Santa Cruz Biotechnology, sc-40) 1:1000; anti-HA-peroxidase (Sigma Aldrich), 1:1500; anti-cyclin A2 (Santa Cruz Biotechnology, sc-596) 1:500; anti-IRS1 (Cell Signaling Technologies, 2382) 1:750; anti-MyoD1 (Cell Signaling Technologies, 13812) 1:750; anti-GAPDH (Abcam, ab8245) 1:2000; anti-α tubulin (Abcam, ab7291 and Santa Cruz Biotechnology, sc-8035) 1:1000 for both; anti-vinculin (Santa Cruz Biotechnology, sc-73614) 1:2000. Secondary antibodies used: anti-rabbit IgG-HRP (GE Healthcare, NA934) and anti-mouse IgG-HRP (GE Healthcare, NA931V), both at 1:3000 dilutions.

Cells were washed three times with cold phosphate-buffered saline (PBS), lysed with NETN lysis buffer (20mM Tris-HCl, pH 7.5; 150mM NaCl; 1 mM EDTA, pH 8.0; 0.5% Nonidet P-40; 1 mM PMSF; 1x protease and phosphatase inhibitors) on ice for 10 minutes and centrifuged for 8 minutes at 12,000 rpm to separate lysates from cell debris. Protein concentrations were determined with BCA Protein Assay Kit (Pierce). Equal amounts of protein from cell lines were loaded and separated on 8–10% SDS-PAGE. Proteins were transferred to Hybond ECL nitrocellulose (Amersham) and blotted using anti-PRKCZ, anti-IGF1R, anti-ITGB3, anti-IRS2 (Cell Signalling), and anti-pIRS2 (Abcam) antibodies at 1:1000 dilutions. Secondary conjugates, HRP-Donkey anti-mouse or HRP-Donkey anti-rabbit (Jacksons Immunochemicals) were incubated for 1 hour at a 1:5000 dilution. Protein bands were visualized by chemiluminescence using ECL detection system (Amersham).