Anti mouse a9917

After separation in SDS-PAGE (AA: 10%, AA/BisAA ratio: 36:1), the proteins were transferred onto PVDF membranes following overnight incubation with specific primary antibodies. The following primary antibodies were used: anti-actin (Sigma A5441, Sigma-Aldrich, St. Louis, MO, USA), anti-Zeb1 (Sigma AMAb90510, Sigma-Aldrich, St. Louis, MO, USA), anti-Nanog (Santa Cruz sc-293121, Dallas, TX, USA), anti-Oct4 (Cell Signaling #2890s, Danvers, MA, USA), anti-Sox2 (Cell Signaling #3579s, Danvers, MA, USA), anti-CTBP2 (Abcam ab128871, Cambridge, UK), anti-CTBP1 (Sigma HPA018987, Sigma-Aldrich, St. Louis, MO, USA), anti-LSD1 (Sigma ABE365, Sigma-Aldrich, St. Louis, MO, USA), anti-TRIM33 (Sigma HPA004345, Sigma-Aldrich, St. Louis, MO, USA), and anti-p53 (Cell Signaling #46565, Danvers, MA, USA). The secondary antibodies were from Sigma: anti-mouse (A9917) and anti-rabbit (A0545). Bound antibodies were visualized using the SuperSignal West Femto Maximum Sensitivity Substrate ECL kit (Thermo scientific, Waltham, MA, USA), chemiluminescence was detected using ChemiDoc (BioRad, Hercules, CA, USA).

10⁷ cells were lysed in 100 µl of RIPA buffer with protease inhibitor cocktail and EDTA (Thermo Fisher Scientific). Protein concentration was determined using Pierce BCA Protein Assay Kit (Thermo Fisher Scientific). Proteins were separated on 4–15% Mini-PROTEAN TGX Stain-free Gel (Bio Rad). After electrophoresis, proteins were transferred onto 0.45 µm PVDF Low Fluorescence membrane (Bio Rad, Hercules, CA, USA). Membranes were blocked using 5% non-fat milk and incubated with 1:2000 mouse anti-Cas9 antibody (#14697, Cell Signaling Technology, Danvers, MA, USA) or 1:2000 rabbit anti-BIM antibody (#2933, Cell Signaling Technology). After washing, membranes were incubated with horseradish peroxidase (HRP) conjugated with 1:10,000 anti-mouse (A9917, Sigma Aldrich) or 1:40,000 anti-rabbit (ab97051, Abcam, Cambridge, U) secondary antibody. Immunoreactive protein bands were detected with Clarity Western ECL Substrate for HRP (Bio-Rad) on Chemidoc Imaging System (Bio Rad). The abundance of target protein was assessed in reference to the total protein on a blot in Stain-Free technology using Image Lab 6.0.1 software (Bio Rad). Each experiment was conducted in three biological replicates.

After separation in SDS–PAGE (AA: 10%, AA/BisAA ratio: 36:1), the proteins were transferred onto PVDF membranes following overnight incubation with specific primary antibodies. The following primary antibodies were used: anti-actin (Sigma A5441, St. Louis, MO, USA), anti-E-cadherin (BD Biosciences, Heidelberg, Germany), anti-Zeb1 (Sigma AMAb90510), anti-CTBP2 (Abcam ab128871), anti-DDX17 (Sigma AV41029), anti-vimentin (Santa Cruz sc6260) and anti-N-cadherin (Cell Signaling 14215S). The secondary antibodies were from Sigma: anti-mouse (A9917) and anti-rabbit (A0545). Bound antibodies were visualized using the SuperSignal West Femto Maximum Sensitivity Substrate ECL kit (Thermo scientific, Boston, MA, USA), chemiluminescence was detected using ChemiDoc Touch Imaging System (Bio-Rad).