Western blotting detection system

RIPA buffer (Thermo Fisher Scientific; Waltham, MA) containing octyl-β-D-glucopyranoside (60 mM, Millipore-Sigma) and protease/phosphatase inhibitor cocktails (Roche, Basel, Switzerland) was used for assessment of Cav-1/Cav-2/Cavin-1 expression. Protein concentration was determined with a protein assay kit (BioRad, Hercules, CA). For albumin immunoblots, two times acid/salt washes were performed on ice for 2 min each, followed by a 1× PBS wash before cell lysis. Proteins were resolved by SDS-PAGE and transferred to nitrocellulose membranes. Membranes were incubated in 5% milk in Tris-buffered saline with Tween (TBST) blocking buffer for 1 h at room temperature. Primary antibodies were allowed to bind overnight at 4°C, and used at a dilution of 1:1,000. After washing in TBST three times for 5 min each, the membranes were incubated with immunofluorescent secondary antibodies at a 1:1,000–5,000 dilution for 1 h at room temperature and developed using western blotting detection system (Thermo Fisher Scientific).

For protein extraction, cells were lysed in lysis buffer containing1% NP-40, 1 mM EDTA, 50 mM Tris-HCl (pH 7.5), and 150 mM NaCl, supplemented with complete protease inhibitor mixture (Roche, Mannheim, Germany). Protein concentration was determined by the Bradford assay (Bio-Rad) using bovine serum albumin as standard. Total proteins were separated on a 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to nitrocellulose membranes (GE Healthcare, Chicago, IL, USA). Membranes were blocked with 5% nonfat dry milk.
After blotting, membranes were incubated with primary antibodies against p21 (DCS60; Cell Signaling Technology, Leiden, Netherlands) or β-actin (sc-1615; Santa Cruz Biotechnology, Dallas, TX, USA). The reaction was detected with a Western blotting detection system (Thermo Fisher Scientific).