Supersignal west femto and pico kits

Cells were lysed with RIPA lysis buffer (25 mM Tris-HCl at pH 7.6, 150 mM NaCl, 1% NP-40 and 0.1% SDS) and centrifuged at 15,000 × g at 4 °C for 15 min. Protein concentration was determined by the BCA assay (Novex®, Life Technologies). Thirty μg of each sample and a PageRuler™ Prestained protein ladder (Thermo Scientific) were loaded on a NuPAGE® 4–12% Bis-Tris gradient gel with 1× NuPAGE® MOPS running buffer (Novex®, Life Technologies) and separated at 150 V for 1 h. Next, a dry transfer was done at 20 V for 6 min using PVDF mini stacks in an iBlot 2 instrument (Thermo Fisher Scientific). The membrane was blocked in 5% dry milk in TBS-T for 30 min and immunoblotted overnight at 4°C with primary antibodies against Cas9 (#14697, CST), p53 (#9282, CST), p21 (#2947, CST), β-actin (sc-47778, Santa Cruz Biotechnology), GAPDH (#5174, CST) and vinculin (V9131, Sigma-Aldrich) diluted 1:1,000 in PBS containing 5% milk. The membrane was washed in TBS-T and further incubated for 1 hour with goat anti-rabbit and goat anti-mouse secondary antibodies (sc-2027 and sc-2025, Santa Cruz Biotechnology) diluted 1:10,000 in TBS-T containing 5% milk. Signal detection was performed with the SuperSignal West Femto and Pico kits (Thermo Scientific) in the ImageQuant LAS 4000 imager (GE Healthcare Life Sciences).