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. 2010 Jul 12;107(30):13444–13449. doi: 10.1073/pnas.0913690107

Fig. 3.

Fig. 3.

Mule ubiquitinates Miz1 in vivo. (A) HEK293 cells were transfected with expression vector encoding Xpress-Miz1, along with HA-Ub (K48O) and/or M2-Mule (WT or the C4341A mutant, 5 μg each), as indicated. Cells were treated with TNFα (5 ng/mL, 15 min) in the presence of MG-132. Ubiquitination of immunoprecipitated Xpress-Miz1 and expression levels of Xpress-Miz1, HA-Ub (K48O), and M2-Mule were determined. (B) HEK293 cells were transfected with control siRNA or siRNA for Mule (100 nM each), followed by treatment with or without TNFα (5 ng/mL, 15 min) in the presence of MG-132. Ubiquitination of immunoprecipitated Miz1 was analyzed by immnoblotting with antiubiquitin antibody. (C–E) HEK293 cells were transfected with Xpress-Miz1 WT (C and D) or the POZ deletion mutant (ΔPOZ) (E), with or without M2-Mule. Cells were treated with or without TNFα (5 ng/mL, 10 min) in the presence of MG-132. The interaction between Xpress-Miz1 and M2-Mule was determined by immnoprecipitation with anti-Xpress antibody, followed by immunoblotting with anti-M2 antibody. (F) HEK293 cells were treated with or without TNFα (5 ng/mL, 10 min) in the presence of MG-132. Interaction between endogenous Mule and Miz1 was determined by immunoprecipitation with anti-Miz1 antibody, followed by immunoblotting with anti-Mule antibody.