Figure 5.
STAMBP stabilizes ULK1 through cleaving its K48-linked ubiquitin chains. (a) Immunoassay of extracts of HEK293 T cells transfected with plasmids for MYC-tagged EV or STAMBP and treated with dimethyl sulfoxide (DMSO; vehicle), MG132 (10 μM) or Baf A1 (0.2 μM) for 6 h. (b) Immunoassay of extracts of HEK293 T cells transfected with plasmid encoding MYC-tagged EV or STAMBP and treated with MG132 for 6 h, followed by denatured IP with anti-ULK1 antibody and immunoblot detection with anti-Ub antibody. (c) Immunoassay of extracts of HEK293 T cells transfected with plasmid encoding MYC-tagged EV or STAMBP and cultured in EBSS medium for the various times (above lane), together with MG132 treatment for 6 h, followed by denatured IP with anti-ULK1 antibody and immunoblot detection with anti-K48-Ub antibody. (d) Purified ubiquitinated ULK1 was incubated with immunopurified Flag-STAMBP in vitro in deubiquitinating buffer. The immunoblot was detected with anti-HA. Data are representative of three independent biological experiments