Figure 7.
OTUD7B modifies the K63-K48 type ubiquitination transition of PIK3C3. (a) Immunoassay of extracts of HEK293 T cells transfected with plasmids for MYC-tagged EV or OTUD7B, and then treated with dimethyl sulfoxide (DMSO; vehicle), MG132 (10 μM), 3 MA (10 mM), CQ (50 μM) or Baf A1 (0.2 μM) for 6 h. (b) Immunoassay of extracts of HEK293 T cells transfected with plasmids encoding MYC-tagged EV or OTUD7B and then treated with MG132 for 6 h, followed by denatured IP with anti-PIK3C3 and immunoblot analysis with anti-Ub. (c) Immunoassay of extracts of HEK293 T cells transfected with plasmids encoding MYC-tagged EV or OTUD7B and then treated with MG132 for 6 h, followed by denatured IP with anti-PIK3C3 and immunoblot analysis with anti-K63-Ub. (d) Purified ubiquitinated PIK3C3 was incubated with immunopurified Flag-OTUD7B in vitro in deubiquitinating buffer. The immunoblot was detected with anti-HA. (e) Immunoassay of extracts of HEK293 T cells transfected with plasmids encoding MYC-tagged EV or OTUD7B and then treated with MG132 for 6 h, followed by denatured IP with anti-PIK3C3 and immunoblot analysis with anti-K48-Ub. Data are representative of three independent biological experiments