Proteasome inhibitor cocktail edta free

Cells were lysed with NP40 (Beyotime Biotechnology, China), and supplemented with proteasome inhibitor cocktail EDTA-free (Roche, Germany) and phosphatase inhibitor cocktail I/II (Topscience, Shanghai, China) 72 h–96 h post-transfection. WCE were collected after centrifugation at 14,000 × g. After protein concentration quantified, WCEs at equal concentration and volume were pre-cleaned by IgG of the same species as the indicated primary antibody and protein A/G agarose. The supernatant was immunoprecipitated over night at 4 °C with indicated primary antibodies and protein for following assays. The precipitated protein complexes were washed using NP40 lysis buffer at least five times before being separated on SDS-PAGE and immunoblotting by the indicated antibodies.
For ubiquitylation immunoprecipitation, cells were transfected by HA tagged ubiquitin (HA-ub) plasmids together with the other indicated plasmids. Before collecting the cell lysate, cells were treated with 20 μmol/L of MG-132 (Topscience, Shanghai, China) for at least 6 h. The following assays were performed as previous described. Antibodies applied in this study were listed in Supplementary Table 4.

Whole cell extract was isolated from 48 h post-transfection cells with RIPA lysis buffer (Beyotime Biotechnology, China) with proteasome inhibitor cocktail EDTA-free (Roche, Germany) and phosphatase inhibitor cocktail I/II (Topscience, Shanghai, China). After protein concentrations determinated by BCA protein assay Kit (Beyotime Biotechnology, China), the proteins were separated by SDS-PAGE, followed by western blotting with indicated antibodies. All the primary antibodies were listed in Supplementary Table 4. HRP conjugated Goat anti-Mouse/Rabbit antibody were used as secondary antibody (Boster, Wuhan, China) for chemiluminescence. Eventually, bands were visualized using SuperSignal™ West Atto Ultimate Sensitivity Substrate (ThermoFisher Scientific, USA) or Immobilob™ Western Chemiluminescent HRP Substrate (Millipore, USA). Antibodies applied in this study were listed in Supplementary Table 4. Uncropped western blot strips are provided in the supplementary material.