Western blotting
LYSED, SECONDARY, LYSIS
Cells were lysed on ice using m-PER lysis buffer (Thermo Fisher Scientific) supplemented with a protease and phosphatase inhibitor (Cell Signaling, 5872) and protein was quantified using Pierce BCA protein assay kit (Thermo Fisher Scientific). In total, 20–50 μg of protein extract was electrophoresed on a 4–15% SDS gradient gel (Mini-PROTEAN TGC Gel, Bio-Rad) and transferred to an activated PVDF membrane for 1 h at 4 °C. Unspecific binding sites were blocked in 5% BSA in Tris-buffered saline with 1% Tween-20 (TBS-T) for 1 h at 4 °C. Primary antibodies were incubated overnight at 4 °C in constant motion in 5% BSA–TBS-T. The mouse anti-Cas9 (S. pyogenes) antibody (Cell Signaling; cat. 14697, clone 7A9-3A3) and rabbit anti-ACSL4 antibody (Abcam; cat. ab155282; clone EPR8640) were used at 1:1,000 dilution in 5% BSA–TBS-T. The mouse anti-actin Ab-5 antibody (BD Biosciences; cat. 612656; clone C4/actin (RUO)) was used at 1:3,000 dilution in 5% BSA–TBS-T. The membrane was washed with TBS-T three times for 5 min before incubation with the secondary antibody for 1 h at room temperature. Polyclonal goat anti-rabbit immunoglobulins/HRP (Dako; cat. P0448) and polyclonal rabbit anti-mouse immunoglobulins/HRP (Dako; cat. P0260) were used at 1:4,000 dilution. After subsequent membrane washing, protein was detected using Immobilon chemiluminescent HRP substrate (WBKLS0500, Millipore) and imaged with the iBright CL1500 imaging system.