Protein lysate quantification

Cells were washed with 1× PBS and lysed on ice with radioimmunoprecipitation assay (RIPA) buffer (50 mM Tris, pH 7.6, 150 mM NaCl, 1% Triton X-100, 0.5% sodium deoxycholate) containing freshly added phosphatase inhibitors (1:100; VWR), protease inhibitors (1:100; Sigma-Aldrich), and 250 nM okadaic acid (1:1,000; Fisher Scientific). Cell lysates were collected and stored at −80°C. Lysates were subsequently thawed on ice and sonicated in a water bath containing ice slurry for three 30-s bursts on the highest setting with two 20- s pauses in between. Lysates were then centrifuged at 14,000 rpm for 15 min at 4°C to pellet the cellular debris. Clarified lysates were collected and quantified using a Bio-Rad protein assay kit (Bio-Rad).