Immunoblotting Protocol for Protein Expression

Immunoblotting was performed as previously described [19 (link),20 (link)]. Briefly, the cells were lysed in a RIPA lysis buffer containing a protease inhibitor cocktail (Sigma-Aldrich, St. Louis, MO, USA) and supernatants were collected by centrifugation at 13,000 rpm for 15 min at 4 °C. Protein quantification in each sample was performed using a BCA™ protein assay kit (Pierce, Rockford, IL, USA) with bovine serum albumin (BSA) as a standard. Equal amounts of proteins were denatured and fractionated by SDS–PAGE, and the proteins were transferred onto nitrocellulose membranes. Nonspecific binding sites were blocked by incubating the membranes in 5% nonfat milk in TBS containing 0.1% Tween-20. The membranes were probed with appropriate primary antibodies overnight at 4 °C followed by appropriate horseradish peroxidase (HRP) conjugated secondary antibody for 1 h at room temperature. Protein expression was detected by enhanced Western Lightning Plus-ECL, enhanced chemiluminescence substrate (PerkinElmer, Shelton, CT, USA). Images were acquired on a Bio-Rad ChemiDoc system by using Image lab software. Expression of GAPDH was used as an internal control for total protein-loading.