Fig. 3: HOIP/OTULIN control the M1 ubiquitination of RTA-K516 and -K518.

a RTA is modified by M1 ubiquitination. RTA-Flag and Ub^KO were co-transfected into HEK293T cells and subjected to immunoprecipitation with anti-Flag magnetic beads. RTA M1 ubiquitination was determined by a specific anti-linear Ub antibody (Genentech). Ub^KO is a ubiquitin mutant that can only form M1 polyUb chains. n = 3 independent experiments. b HOIP/HOIL-1L promotes RTA M1 ubiquitination. RTA-Flag and Myc-HOIP/Myc-HOIL-1L were co-transfected into HEK293T cells. RTA-Flag was immunoprecipitated with anti-Flag magnetic beads and detected with anti-linear Ub and RTA antibodies, respectively. n = 3 independent experiments. c OTULIN reduces RTA M1 ubiquitination. As in (b), but the plasmids were replaced with the indicated plasmids. n = 3 independent experiments. d Endogenous RTA displays M1 ubiquitination. iSLK/rKSHV.219 cells were transfected with Myc-OTULIN or Myc-OTULIN^C129A and then treated with Tet plus VPA. RTA was immunoprecipitated with anti-RTA antibody and immunoblotted with anti-linear Ub and anti-RTA antibodies, respectively. n = 3 independent experiments. e Diagrams of RTA domains, the amino acid sequence of the RTA NLS2 and the Lysine (K) to Arginine (R) mutants used in the study. f RTA^2KR is defective in M1 ubiquitination. HEK293T cells were transfected with plasmids as indicated and cell lysates were immunoprecipitated using anti-Flag beads. Immunoprecipitated proteins were immunoblotted with the indicated antibodies. n = 3 independent experiments. Source data are provided as a Source Data file.