Immunoprecipitation and Ubiquitination Assay Protocol

For immunoprecipitation, Aag2 cells were transfected with indicated plasmids and suspended in lysis buffer containing 50 mM Tris-HCl (pH 7.5), 1% Nonidet P40, 0.5% sodium deoxycholate, 150 mM sodium chloride, 1 tablet complete protein inhibitor cocktail/50 mL, and 0.7 μg/mL pepstatin. To reduce the nonspecific binding of unrelated proteins to protein A-agarose (Roche), a 50-μL suspension of homogeneous agarose beads was mixed with the sample, incubated at least 3 h at 2°C to 8°C, and then centrifuged for 20 s at 12,000 × g. The supernatant was separated and incubated with specific antibodies at 4°C overnight. The beads with complex were pulled down and washed with lysis buffer, washing buffer 2, and washing buffer 3, according to the producer’s instructions. After centrifugation, the agarose beads were resuspended in 2× SDS loading buffer, and proteins were heat denatured. After removal of the agarose, the mixture was subjected to immunoblotting with different antibodies (all antibodies used are listed in Table S6). Ubiquitination assays were performed as described elsewhere (53 (link)). After homogenizing in lysis buffer, the tissue samples were centrifuged. The supernatant was mixed with SDS sample buffer and then heated before immunoblotting with anti-ubiquitin antibodies.