Whole cell lysate extraction and subcellular fractionation

To collect whole cell lysate, cells were lysed in RIPA buffer (25 mM Tris-HCl [pH 7.4], 0.1% SDS, 1% NP-40, 0.5% sodium deoxycholate, 150 mM NaCl, protease inhibitor cocktail, 1 mM Na₃VO₄, and 1 mM NaF). For subcellular fractionation, cells were trypsinized and centrifuged to collect the cell pellet. The cell pellet was incubated with Lysis Buffer A (50 mM HEPES [pH 7.4], 150 mM NaCl, 1 M hexylene glycol, 25 μg/mL digitonin, protease inhibitor cocktail, 1 mM Na₃VO₄, and 1 mM NaF) on ice for 10 min to extract the cytosolic proteins. The cell pellet was washed with Lysis Buffer A and was then incubated with Lysis Buffer B (50 mM HEPES [pH 7.4], 150 mM NaCl, 1 M hexylene glycol, 1% v/v Igepal, protease inhibitor cocktail, 1 mM Na₃VO₄, and 1 mM NaF) on ice for 30 min to extract the membrane-bound proteins. The cell pellet was then washed twice with Lysis Buffer B, after which it was incubated with Lysis Buffer C (50 mM HEPES [pH 7.4], 150 mM NaCl, 1 M hexylene glycol, 0.5% w/v sodium deoxycholate, 0.1% w/v sodium dodecyl sulfate, protease inhibitor cocktail, 1 mM Na₃VO₄, and 1 mM NaF) and benzonase to extract the nuclear proteins.