Hrp andsupersignal west pico chemiluminescent substrates

Samples were homogenized in RIPA lysis buffer (Thermo Scientific, Rockford, IL) supplemented with a protease inhibitor cocktail (Roche, Mannheim, Germany). Following centrifugation at 12,000 rpm for 20 min at 4 °C, soluble supernatant fractions were collected for total protein and western blot analysis. Total protein concentrations were determined by BCA assay kit (Thermo Scientific, Rockford, IL). Ten micrograms (μg) total protein were resolved in stain-free, pre-cast 4–15% gradient Tris–Glycine gel (Bio-Rad, Hercules, CA), and stained with primary antibodies of keratin 5 (KRT5; 1:2000, Biolegend, San Diego, CA), integrin beta 4 (ITGβ4, CD104; 1:1000, Invitrogen, Waltham, MA), and plectin (PLEC; 1:1000, ThermoFisher, Waltham, MA). Gels were transferred to 0.2 µm nitrocellulose membrane (Pall Corporation, NY). HRP and SuperSignal West Pico chemiluminescent substrates (Thermo Scientific, Rockford, IL) were used to detect protein signal intensity. Image Lab software (Bio-Rad, Hercules, CA) were used for target protein normalization and quantification. Full length blots were included in the online supplement.