Phosphorylase inhibitor cocktail

Cells were washed three times with ice-cold PBS and then lysed in lysis buffer containing 1 mM phenylmethylsulfonyl fluoride, 1% (v/v) protease inhibitor cocktail, 1% (v/v) phosphorylase inhibitor cocktail, and 1 mM dithiothreitol (all from Sigma, St. Louis, MO, USA). Nuclear and cytoplasmic protein was isolated by using a Nuclear and Cytoplasmic Extraction Kit (Thermo Scientific, West Palm Beach, FL, USA). Cell supernatants were mixed with methanol and chloroform and centrifuged in 4 °C to precipitate the protein, and then protein pellets were dissolved in lysis buffer. Equal amounts of cell lysates were resolved by SDS-PAGE and then transferred to polyvinylidene difluoride membranes. The membranes were blocked in 5% Bovine Serum Albumin PBST (pH 7.4 PBS, 0.5% Tween20) at room temperature for 1 h and incubated at 4 °C overnight with the respective primary antibodies, followed by a second incubation at room temperature for 1 h with appropriate HRP-conjugated secondary antibodies. Finally, blots on the membranes were visualized with Plus-ECL (KeyGEN, Nanjing, China) according to the manufacturer’s protocol.

Cells were lysed with cell lysis buffer containing 1mM phenylmethylsulfonyl fluoride, 1% protease inhibitor cocktail, 1% phosphorylase inhibitor cocktail, and 1 mM dithiothreitol (all from Sigma Aldrich, St. Louis, MO, USA). Cell lysate samples were boiled, separated on SDS-PAGE, and then transferred to PVDF membranes. Membranes were blocked with 5% (w/v) nonfat milk and incubated with a primary antibody overnight at 4°C, followed by a second incubation at room temperature for 1-2 h with appropriate HRP-conjugated secondary antibodies. After further washing with PBST, blots on the membranes were visualized with ECL reagent (KeyGEN, Nanjing, China) according to the manufacturer’s protocol. Equal protein loading was confirmed in all the experiments by using GAPDH or β-actin as loading control.