Precast tris hcl gels

Protein concentrations were measured using the BCA Protein Assay Kit. For SDS/PAGE, cell lysates containing the same amount of total proteins were loaded onto 4–20% precast Tris-HCl gels (Bio-Rad). Proteins were transferred to PVDF membranes (Millipore). All antibodies were diluted in TBST [50 mM Tris (pH 8.0), 150 mM NaCl, and 0.1% (vol/vol) Tween 20] containing 5% (wt/vol) BSA. secondary antibodies were horseradish peroxidase-conjugated mouse anti-goat, mouse anti-rabbit, or mouse IgG Fc binding protein (1:5,000, Santa Cruz, USA). Enhanced chemiluminescence (ECL) was detected using SuperSignal West Pico chemiluminescent substrate (Pierce, USA). Uncropped blots for the main and supplementary figures are provided at the end of supplementary information file.

The cell lysate (50 μl) was diluted with 50 μl of 2× Laemmli buffer (Shapiro et al., 1967 (link)) (52.5 mM Tri–HCL, pH 6.8, 25% glycerol, 2% SDS, 0.02% bromophenol blue) with or without the addition of 150 mM DTT and heated at 95°C for 10 min. Subsequently, the lysate was centrifuged at 12,000 × g in a micro-centrifuge for 5 min and the proteins (50 μl) in the supernatant were separated by SDS–PAGE using the Mini-PROTEAN electrophoresis system (Bio-Rad Laboratories, Hercules, CA, United States) as described by manufacturer’s insert at a constant current of 30 mAmp per gel, using 4–20% precast Tris–HCL Gels (Bio-Rad Laboratories, Hercules, CA, United States). The gels were stained overnight with Imperial protein stain (Thermo Fisher Scientific, Rockford, IL, United States) as per the manufacturer’s protocol. The gels resolved in reducing and non-reducing conditions were imaged using the Kodak Image Station 2000R (Carestream Health). The images for gels were visually compared for missing bands in the reduced gels that were targeted for excision and protein identification as using LC–MS/MS at the Center for Integrated BioSystems, Utah State University (Logan, UT, United States) (Liang et al., 2006 (link); Supplementary Figure S1).