Any kd criterion tgx precast gels

Total protein concentration was normalized according to BCA assay prior to western blot unless stated otherwise. Thereafter, samples were denatured by heating to 95 °C for 5 min in Laemmli sample buffer containing 10% β-mercaptoethanol, and run on AnykD™ Criterion™ TGX™ precast gels (Bio-Rad Laboratories) with tris-glycine-based running buffer. Proteins were transferred from polyacrylamide gels onto polyvinylidene difluoride (PVDF) membranes using a semi-dry transfer system (Trans-Blot^® Turbo™ System, Bio-Rad). Non-specific binding sites were blocked with 5% (w/v) skimmed milk in Tris-buffered saline with 0.05% (v/v) Tween20 (TBST) for 1 h and the membrane was incubated at 4 °C overnight under gentle shaking with the primary antibody in TBST with 5% (w/v) BSA (Cell Signaling Technology). The membrane was washed and incubated with the respective HRP-conjugated secondary antibody (Vector Labs) in TBST for 2 h at room temperature. The protein bands were detected by using Clarity™ Western ECL Substrate Kit (Bio-Rad Laboratories) or ECL Prime™ (GE Healthcare), and LI-COR Odyssey^® Fc Imaging system (LI-COR Biosciences). Band intensities were quantified using Image Studio™ software (LI-COR Biosciences). For proteins of interest, band intensities were normalized to the housekeeping protein GAPDH. All antibodies used for western blot are listed in Supplementary Table 2.