Ubiquitination Assay for Protein Degradation

Ubiquitination assay was performed as previously described [58 (link)]. GSCs overexpressing shNT or shPML shRNAs were treated with the proteasome inhibitor MG132 (20 μM, Sigma-Aldrich, C2211–5MG) for 6 hours and then lysed in TritonX-100 lysis buffer, immunoprecipitated with anti-Myc agarose affinity gel (Sigma-Aldrich, A7470–1ML) and immunoblotted with an anti-ubiquitin (Biolegend, 646301) or anti-c-Myc antibody (Santa Cruz, sc-40). Briefly, cell lysates (300 μg of total protein) were incubated with 15 μL anti-Myc conjugated agarose gel with constant rotation overnight at 4°C. Immunocomplexes were washed three times with ice-cold 0.3% Triton X-100 in PBS buffer and eluted in loading buffer by boiling for 10 min, and then analyzed by immunoblotting. Proteins were resolved on NuPAGE Novex 4–12% Bis-Tris gels (Invitrogen, NP0322BOX), blotted onto polyvinylidene membranes and probed by antibodies specific to ubiquitin and c-Myc.