Laemmli 6x sds sample buffer

Tissue samples were crushed on dry ice to obtain homogenous aliquots and then lysed in RIPA buffer (Thermo Scientific #89900) containing Halt protease & phosphatase inhibitor (Thermo Scientific #1861280). Extracted proteins were quantified using the Pierce BCA Protein Assay Kit (Thermo Scientific #23225). 75 µg of protein lysates were heated for 10 mins at 95°C in Laemmli 6X SDS sample buffer (Boston BioProducts, # BP-111R) with 5% 2-mercaptoethanol (VWR Life Science #M131–100ml) and then ran on 4–20% Criterion TGX pre-cast gels (Bio-Rad #5671093). Separated proteins were transferred onto Immobilon-P Transfer membranes (MilliporeSigma #IPVH00010), blocked for an hour using 5% milk in TBST, and stained overnight with targeting primary antibodies in 5% milk TBST. Stained membranes were then washed with TBST and stained with corresponding secondary antibodies in 5% milk in TBST for one hour. Antibody-stained protein signal was amplified and visualized using SuperSignal^™ West Pico PLUS chemiluminescent substrate (Thermo Fisher #34578) and imaged with a ChemiDoc MP Imaging system (Bio-Rad). The antibodies used for the immunoblot assays were: GCLC (Santa Cruz Biotech, #sc-390811), NQO1 (Sigma Prestige Antibodies, HPA007308), ACTIN (Sigma, A1978).