Fig. 3

OTUB1 stabilizes UBC13 by reducing its K48-linked polyubiquitination. a–e Cytoplasmic proteins from unstimulated, TgPFN-stimulated (a, b, e) and LPS-stimulated (c, d) BMDCs were immunoprecipitated with the indicated antibodies. Immunoprecipitates and input were analyzed by WB analysis with the indicated antibodies. f OTUB1-sufficient and OTUB1-deficient FLT3L-expanded BMDCs were pretreated with TgPFN (1 μg/ml) for 1 h or left unstimulated. Then, cycloheximide (CHX, 100 μg/ml) was added for the indicated time period. Protein levels of UBC13 in whole-cell lysates were analyzed by WB analysis (upper panel). The lower panel shows the relative levels of UBC13 normalized to βACT levels (n = 3). g NIH 3T3 cells were transfected with OTUB1 siRNA for 36 h. Then, the cells were transfected with GFP, GFP-OTUB1, GFP-OTUB1 ΔN, or GFP-OTUB1 C91S plasmids. After 24 h, the cells were treated with CHX + LPS for 0, 3, and 6 h. Whole-cell lysates were then isolated and analyzed with the indicated antibodies. h–k GM-CSF-expanded BMDCs were left unstimulated or stimulated with TgPFN in the presence of MG132. Cytoplasmic proteins were isolated and immunoprecipitated with anti-TRAF6 (h, i) and anti-IRAK1 (j, k) antibodies. Immunoprecipitates and input were analyzed with the indicated antibodies. l FLT3L-expanded BMDCs were left untreated or transduced with UBC13-expressing lentivirus or vector lentivirus for 72 h, followed by stimulation with TgPFN for 24 h. Cytokines in the supernatants of cell cultures were measured by flow cytometry (n = 4). Data are displayed as the mean ± SD (d) or mean ± SD (g). *p < 0.05, **p < 0.01, and ***p < 0.001; n.s. not significant