Mouse flag mab m2

Protein samples were isolated by resuspending cell pellets in RIPA buffer (50 mM Tris–HCl at pH 7.6, 150 mM NaCl, 1% NP-40, 0.5% Na deoxycholate, 0.1% SDS). After removal of the debris, samples were quantified with colorimetric BCA kit (Pierce). 20 μg total proteins were electrophoresed on 6–12% gradient gels and wet-transferred to nitrocellulose membranes. After 1 h blocking with 5% nonfat dry milk in 1 × TBS, 0.1% Tween20 at room temperature, membranes were incubated with antibodies diluted in 2% w/v BSA as follows; Flag [M2] mouse mAb (1:1000, Sigma) to detect DD-SpCas9 expression, α-tubulin [DM1A] mouse mAb (1:20,000, Millipore) as equal loading control, GFP [C163] mouse mAb (1:5,000, Thermo Fisher) to detect Venus expression, p53 [DO-1] mouse mAb (1:1,000, Millipore), CypD [E11AE12BD4] mouse mAb (1:5,000, Abcam), RPA14 [11.1] mouse mAb (1:1,000, Abcam), EGFR [1F4] mouse mAb (1:1,000, Cell Signaling Technology) and DD monoclonal Ab (1:1,000, Clontech). All incubations were performed overnight at 4 °C. Membranes were rinsed thoroughly with 1 × TBS-T and then incubated with species-specific fluorescently labelled secondary antibodies (1:5,000, LICOR). Western blots were eventually imaged on near-IR fluorescence scanner (Odyssey Imaging System, LICOR).