Image laboratory version 5

A detailed explanation of the Western blot analysis protocol has been published elsewhere54 (link). Equal amounts (20 μg) of protein for each treatment group were resolved on 4–15% TGX stain-free gels (Bio-Rad Laboratories GmbH, Munchen, Germany) and transferred onto PVDF membranes. The membranes were probed with the primary antibody in 0.01 M Tris-buffered saline supplemented with Triton X-100 (TBST) containing 5% nonfat dry milk followed by HRP-conjugated secondary antibody. When necessary, PVDF membranes were stripped using Restore PLUS Western blot stripping buffer (Thermo Scientific, Rockford, IL) for 15 minutes at room temperature, washed twice in TBST, and then reprobed. Ultraviolet activation of the Criterion stain-free gel on a ChemiDoc MP Imaging System (Bio-Rad) was used to control for proper loading21 (link). Band densitometry was performed using Image Laboratory (Version 5.0, Bio-Rad).

Liver and adipose tissues were lysed using RIPA buffer (Sigma-Aldrich) supplemented with cOmplete Mini protease inhibitor cocktail tablets (Roche Diagnostics, Mannheim, Germany) and PhosSTOP phosphatase inhibitor cocktail tablets (Roche Diagnostics). Equal amounts (20 μg) of protein for each treatment group were resolved on 4–15% TGX stain-free gels (Bio-Rad Laboratories GmbH, Munich, Germany) and transferred onto PVDF membranes. The membranes were probed with the primary antibody (Supplementary Table 1) in 0.01 M Tris-buffered saline supplemented with Triton X-100 (TBST) containing 5% nonfat dry milk followed by HRP-conjugated secondary antibody. When necessary, the PVDF membranes were stripped using Restore PLUS Western blot stripping buffer (Thermo Scientific, Rockford, IL) for 15 minutes at room temperature, washed twice in TBST, and then reprobed. Ultraviolet activation of the stain-free gel on a ChemiDoc MP Imaging System (Bio-Rad) was used to control for proper loading [19 (link), 59 (link)]. Blots were scanned and quantified using Image Laboratory (Version 5.0, Bio-Rad, Sweden). All specific protein band densities were normalized to the total-protein loading control.