Enhanced chemiluminiscence

Protein extracts from islets and MIN6 transfected cells were prepared in lysis buffer (50 mmol/l Tris pH 7.5, 5 mmol/l EDTA, 150 mmol/l NaCl, 1% Triton X-100, 10 mmol/l sodium phosphate) containing fresh protease and phosphatase inhibitor cocktails (Roche, Basel, Switzerland). Proteins were separated by 8% SDS-PAGE and transferred to nitrocellulose membranes. Immunoblots were performed using the following antibodies: rabbit monoclonal anti-PTP1B antibody (Novus Biologicals, Littleton, CO, USA), rabbit anti-STAT3 antibody, rabbit anti-pSTAT3^Tyr705 antibody, rabbit anti-AKT IgG, rabbit anti-pAKT^Thr308 antibody, rabbit anti-ERK1/2 (p44/42 MAPK) antibody, rabbit anti-pERK^{Thr202/Tyr204} antibody, rabbit anti-FOXO1 IgG, rabbit anti-p FOXO1^Ser256 antibody (all of them from Cell Signaling) and mouse monoclonal anti-p53 antibody (ABCAM, Cambridge, UK). As a loading control, rabbit anti-actin antibody was used (Sigma Aldrich). The antibody dilution used was 1∶1000.
Immunoblots were developed with horseradish peroxidise-conjugated secondary antibodies (GE Healthcare Bio-Sciences Corp. Piscataway, NJ, USA) and visualized using enhanced chemiluminiscence (Thermo Fisher Scientific Inc. Waltham, MA, USA). Bands were detected by an ImageQuant LAS 4000 camera (GE Healthcare) and quantified by densitometry scanning with Image J software.

Western blotting was performed as described36 (link) using unstimulated and stimulated cells with IL 17A after 6 h of stimulation. All the primary antibodies used for this work were obtained from commercial sources. SREBP2, SREBP1, ACACA and HMGCR from Abcam (Cambridge, MA, USA) and FASN and GAPDH Santa Cruz (Santa Cruz Biotechnology, Santa Cruz, CA, USA). The secondary antibodies were HRP-linked and blots were developed using enhanced chemiluminiscence (Thermo Scientific, Waltham, MA, USA).