Immuno blot pvdf membranes

Proteins were extracted from cultured cells using Lysis Buffer (50 mM Tris-HCl, pH 7.5; 150 mM NaCl; 2 mM EDTA; 1% IGEPAL CA-630; and 0.5% Triton X-100) supplemented with protease inhibitors at 4°C. Cell lysates were spun at 11,000g for 30 minutes at 4°C and the supernatants contained the detergent-soluble fraction. The pellets containing the detergent-insoluble fractions were resuspended using the Lysis Buffer. Both the detergent-soluble and -insoluble protein samples were size fractionated on Novex 4-20% Tris-Glycine Mini Protein Gels (Thermo Fisher Scientific: XP04200BOX) and transferred overnight onto Immuno-Blot PVDF membranes at 4°C. The membranes were blocked for 30 minutes at room temperature using 3% Blotting-Grade Blocker in 1X PBST, and incubated with indicated antibodies overnight with rotation/nutation at 4°C. The following day, the membranes were washed in 1X PBST, incubated with indicated secondary antibodies for 2 hours at room temperature and proteins were detected using ECL Western blotting detection reagents.

Whole muscle lysate was extracted by homogenizing 5mg of gastrocnemius muscle piece from each sample in RIPA buffer (ThermoFisher, 89901). A total of 20μg protein per sample was separated on a 4–12% SDS-polyacrylamide gel with MOPS SDS Running Buffer (Novex-Life Technologies) at 100V. The protein was then transferred onto Immuno-Blot PVDF Membranes using the iBlot2 Dry Blotting System (Thermo Fisher Scientific). After 1h of blocking (Life Technologies) at room temperature, the membranes were incubated with 1:1000 anti-PGC-α antibody (#2178, Cell Signaling) at 4°C overnight on slow rocking, followed by a 1h incubation with 1:1000 anti-rabbit HRP-conjugated secondary antibody (#G-20234, ThermoFisher) at room temperature. Each incubation was followed by three rounds of 5-minute-long washing with TBS-T buffer. For immunodetection, the membranes were immersed in ECL solutions per manufacturer’s specifications (Amersham Biosciences) and exposed to Hyperfilm. For GAPDH detection, the same membranes were incubated with 1:1000 anti-GAPHD (#2118, Cell Signaling) after washing. Band intensities on the films were analyzed using ImageJ software.