Ab4812

For exogenous immunoprecipitation, after 48 hours of expression plasmid transfection into HEK293FT cells, cells were collected and lysed in a chilled nuclear lysis buffer (50 mmol/L Tris‐HCl (pH 8.0), 150 mmol/L NaCl, and 1% Nonidet P40) with 2% (v/v) complete proteinase inhibitor (CPI) and NaVO₃. Cells were sonicated, and the supernatants were collected by centrifugation at 14 000 g for 15 minutes at 4°C. Flag‐conjugated protein A + G agarose beads (Sigma) were added to the samples; afterwards, the samples were incubated for 7‐9 hours at 4°C with gentle rotation. For endogenous immunoprecipitation, whole‐cell lysate of abundant U251 cells (about 1 × 10⁷) in the nuclear lysis buffer was divided equally into 2 microcentrifuge tubes and incubated with anti‐USP22 antibody (ab4812; Abcam) and anti‐IgG antibody (ab109489; Abcam) at 4°C respectively. After 6 hours, non‐tagged protein A + G beads (Santa Cruz Biotechnology) were added to each microcentrifuge tube and the mixed samples were incubated at 4°C overnight. After that, the beads were precipitated by centrifugation at 8000 g for 30 seconds at 4°C. Then, the beads were washed 4 times with 1 mL ice‐cold lysis buffer, and the immunoprecipitated proteins were released from the beads by boiling the sample buffer for 2 minutes. The proteins were detected by western blot assay as previously mentioned.