FLAG-OTUB1 Deubiquitinase Assay

1.5 x 10⁸ HEK293 cells stably expressing empty vector (control), FLAG-OTUB1 WT or N22A were harvested by trypsinisation, washed 1x in cold PBS and lysed in 1 ml 1x TBS plus 1% NP40, 5 mM MgCl₂, 1 mM PMSF and 1x Roche cOmplete EDTA-Free Protease Inhibitor Cocktail per sample on ice for 30 min. Lysates were clarified by centrifugation at 14,000 rpm at 4°C for 10 min and pre-cleared by incubating with 12.5 μl Sepharose 6 fast flow resin and 12.5 μl Protein G Dynabeads (prepared according to manufacturer’s instructions) for 30 min at 4°C with rotation. Immunoprecipitation was performed on pre-cleared lysates by adding 25 μl Protein G Dynabeads and 10 μl ANTI-FLAG M2 antibody per sample and incubating for 2.5 h at 4°C with rotation. Beads were then washed 3x in cold TBS + 0.2% NP40 with a final wash in cold TBS before resuspending in 60 μl TBS. Immunoprecipitated FLAG-OTUB1 WT and N22A (bound to Protein G Dynabeads, equivalent to approximately 3 x 10⁷ cells) was incubated with 600 nM K48-tetraubiquitin (K48-Ub₄) at 37°C. Samples were harvested at 0, 30, and 60 min. Empty vector IP beads were used as a negative control and 26S proteasomes as a positive control for DUB activity. Samples were mixed with SDS loading buffer, heated at 90°C for 5 min and separated on 4%–12% bis-tris gels. Proteins were transferred to PVDF membranes and blocked in PBS/0.2% Tween-20 plus 3% BSA for 1 h at room temperature. Proteins were detected with HRP-conjugated P4D1 anti-ubiquitin antibody and. ANTI-FLAG M2 antibody.