Protease and phosphatase inhibitor cocktails for cell lysis

Cerebral organoids were harvested at pre-defined time points and lysed with RIPA Lysis and Extraction Buffer supplemented with Protease and Phosphatase Inhibitor Cocktails for Cell Lysis (Roche). Lysed samples were sonicated and incubated for 60 min on ice. Samples were centrifuged in an ultracentrifuge (Beckman–Coulter) at 100,000 × g, for 1 h at 4 °C. Supernatants (soluble fraction) were collected and the pellet was resuspended in 50 μl of 70% formic acid (FA), sonicated, and neutralized with 2.5 M Tris buffer (pH 8.5) (insoluble fraction). Total protein concentration in the soluble fraction was determined using a Pierce BCA Protein Assay Kit.

Cerebral organoids or cells were lysed with RIPA Lysis and Extraction Buffer supplemented with Protease and Phosphatase Inhibitor Cocktails for Cell Lysis (Roche). Samples were kept on ice for 60 min after sonication, and then centrifuged in an ultracentrifuge (Beckman-Coulter) at 100,000 g, for 1 h at 4 °C. Supernatants were collected for Western blotting analysis. Total protein concentration in the soluble fraction was determined using a Pierce BCA Protein Assay Kit.