Anti ubiquitin

Mouse CD43⁻ resting B cells, which were stimulated with 1 μg/ml anti-mouse IgM Ab and 100 μg/ml poly(I:C) for 72 h, were lysed in RIPA buffer (50 mM Tris-HCl, 150 mM NaCl, 0.1% SDS, 0.5% sodium deoxycholate and 1% Nonidet P-40) in the presence of a protease inhibitor cocktail (Sigma-Aldrich). After 30 min incubation, the cells were sonicated by ultrasonication (Emerson Electric, St. Louis, MO, USA). Supernatants were collected after centrifugation at 15,000 g for 30 min and mixed with SDS sample buffer with 2-mercaptoethanol (Sigma-Aldrich). After 5 min boiling, the proteins were separated by NuPAGE 4–12% Bis-Tris gel electrophoresis (Invitrogen) and transferred onto polyvinylidene fluoride (PVDF) membrane (Merck Millipore). Anti-IRF4 (1:1,000 dilution, BioLegend), anti-IRF5 (1:1,000 dilution, Abcam, Cambridge, MA, USA), anti-IRF8 (1:1,000 dilution, Abcam), anti-ubiquitin (1:2,000 dilution, BioLegend), and anti-β-Actin antibodies (1:4,000 dilution, BioLegend) were used to detect the specific proteins. Chemiluminescence was developed using ECL Select Western Blotting Detection Reagent (GE Healthcare Life Sciences, Pittsburgh, PA, USA) and exposed to the LAS-3000 Mini Imaging System (FUJIFILM, Tokyo, Japan).

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.