Thermo scientific supersignal west femto chemiluminescent substrate

CFTR: FRT and 16HBEge cells were washed with PBS, harvested, and lysed with ice-cold RIPA buffer containing 1% EDTA and 1% protease inhibitors (Halt Thermoscientific protease inhibitor cocktail, Catalog # PI78425, Thermo Fisher). Protein concentrations were determined using the Pierce^TM BCA Protein Assay Kit (Catalog # PI23250, Thermo Fisher). Monoclonal CFTR primary antibody (UNC 596; 1:5000; received under MTA from the University of North Carolina at Chapel Hill) and mouse monoclonal anti-α-tubulin (1:5000; Catalog # 62204, Thermo Fisher) antibodies were used to detect CFTR and α-tubulin levels, respectively. Protein bands were visualized using Thermo Scientific™ SuperSignal™ West Femto Chemiluminescent Substrate (Catalog # PI34096, Thermo Fisher) for CFTR proteins or Thermo Scientific™ SuperSignal™ West Pico PLUS Chemiluminescent Substrate (Catalog # PI34579, Thermo Fisher) for α-tubulin. Images were captured by a Gel Doc XR + Gel Documentation System (Bio-Rad).

The western blot analysis was performed as previously described [60 (link)]. In brief, cells were lysed with Cell Lysis Buffer (Cell Signaling) containing Protease Inhibitor Cocktail (Sigma). Total proteins were separated by a 4–20% Criterion TGX Precast Gel (Bio-rad) and then transferred onto Trans-Blot® Turbo™ Midi Nitrocellulose Transfer Packs (Bio-rad). The membrane was incubated with primary followed by secondary antibodies after blocking with 5% non-fat milk (Bio-rad). Immunochemical detection was performed using either the Thermo Scientific™ SuperSignal™ West Femto Chemiluminescent Substrate (Thermo Scientific) or Amersham™ ECL™ Western Blotting Detection Reagent (GE Healthcare Life Science). The following antibodies were used at the dilution recommended by the manufacturer: β-actin (Sigma Aldrich, AC-15), GAPDH (Santa Cruz, sc-51905), XIAP (Cell Signaling, #2042), PARP (Cell Signaling, #9542), Survivin (Cell Signaling, 6E4), c-IAP (Cell Signaling, #4952), ZEB1 (Santa Cruz, sc-10572), and vinculin (Cell Signaling, E1E9V).