Ubiquitin Pulldown Assay in 293T Cells

The assay was performed as described previously [13 (link)]. Briefly, HA-tagged ubiquitin and the desired constructs were co-transfected into 293T cells. Thirty-six hours post-transfection, cells were treated with 10 μM MG132 for overnight. Cells were collected and lysed in 200 μL denaturing buffer (1% SDS, 50 mM Tris pH 7.5, 0.5 mM EDTA and 1 mM dithiothreitol). After incubation at 100 °C for 10 min, the lysate was sonicated and diluted tenfold with EBC lysis buffer and incubated with anti-HA-conjugated agarose beads (Sigma, mouse antibody) for 4 h at 4 °C. Immunoprecipitants were washed five times with NETN buffer before they were resolved by SDS-PAGE and immunoblotted with the indicated antibodies.