Anykd gradient

Total protein lysates were extracted from NFASC silenced and control cells using Mammalian Cell & Tissue Extraction kit (BioVision, Milpitas, CA) and membrane fractions were isolated using Mem‐PER™ Plus Membrane Protein Extraction Kit (ThermoFisher Scientific). Concentrations were determined by Reducing Agent Compatible BCA Protein Assay kit (Biovision). Total protein lysates (80 μg) or membrane fractions (15 μg) were separated by 10% or AnyKD gradient (Biorad, Hercules, CA) SDS‐PAGE as appropriate and transferred to an Immobilon PVDF membrane (Millipore, Bedford, MA). To prevent non‐specific background binding, the membranes were incubated with 5% non‐fat milk in Tris‐buffered saline with 0.05% Tween‐20 (TBST) for 1 h at room temperature. Membranes were incubated with primary antibodies against p‐AKT, AKT, p‐ERK, ERK, p‐STAT1, STAT1 p‐STAT3, STAT3, (Cell Signaling Technology, Inc., Beverly, MA), NFASC (Origene, Rockville, MD), and GAPDH (Santa Cruz Biotechnology, Santa Cruz, CA) over night at 4°C. After washing three times with TBST, the membranes were incubated with HRP‐conjugated secondary antibody (Cell Signaling Technology) for 1 h at room temperature and proteins visualized by SuperSignal West Pico Chemiluminescent Substrate (ThermoFisher Scientific).