Plus ecl

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.

Western blot was performed as described previously52 (link)53 (link)54 (link). In brief, equal amounts of cell lysates were resolved by SDS-PAGE and then transferred to polyvinylidene fluoride or nitrocellulose membranes. Membranes were blocked and incubated overnight with primary antibodies at 4 °C. The membranes were incubated with appropriate HRP-conjugated secondary antibodies at room temperature for 1 h, and the blots were visualized with PlusECL (KeyGEN BioTECH) according to the manufacturer’s protocol. Alternatively, blots were detected with IRDye 800 CW conjugated anti-rabbit IgG or IRDye 680 CW conjugated anti-mouse IgG secondary antibodies (LI-COR Biosciences, Lincoln, NE), and visualized using Odyssey infrared imaging system (LI-COR Biosciences).