Chemidoc image device

Twenty‐five milligrams of muscle biopsies were homogenized with RIPA lysis buffer (Millipore, Temecula, CA) with added Halt™ protease and phosphatase inhibitor cocktail (Thermo Fisher Scientific, MA). After centrifugation, supernatants were collected and total protein concentration was determined using the Pierce™ BCA Protein Assay Kit (Thermo Fisher Scientific). Equal amounts of protein were boiled in Laemmli buffer at 95°C for 5 min. 20 μg of protein was separated by SDS‐PAGE and transferred to PVDF membranes (Bio‐Rad Laboratories, Inc., CA) using the semidry Trans‐Blot Turbo™ device (Bio‐Rad). Membranes were incubated with the following primary antibodies, total p62, Sestrin1 and 3 (Abcam, ab56416, ab103121, and ab97792, respectively), Sestrin2 (ProteinTech, 10795‐1‐AP), and p62^Ser403 (GeneTex, GTX128171) (all at 1:1000 dilution, except Sestrin1 which is at 1:100) overnight, and the appropriate anti‐rabbit or anti‐mouse secondary antibodies (Jackson ImmunoResearch Laboratories, PA) linked to horseradish peroxidase (1:10,000) for 1 h at room temperature. The membranes were exposed on a ChemiDoc image device (Bio‐Rad) using enhanced chemiluminescence reagent (ECL Select kit; GE Healthcare Ltd., Little Chalfont, UK). Bands were quantified using ImageJ software (NIH, Bethesda, MD). Western blot data were normalized to the housekeeping protein GAPDH (Abcam, ab36840) (1: 10,000).