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. 2011 Dec 19;109(1):191–196. doi: 10.1073/pnas.1105176108

Fig. 4.

Fig. 4.

Miz1 is polyubiquitinated at Lys388 and Lys472 upon TNF-α stimulation. (A) HeLa cells transfected with expression vectors encoding Xpress-Miz1, along with HA-Ub or empty vector (5 μg each) were treated with TNF-α (5 ng/mL, 15 min) and harvested in the presence or absence of deubiquitination enzyme (DUB) inhibitors (Ub aldehyde 5 μM and N-ethylmaleimde 8 mM). CBB, coomassie brilliant blue. (B and C) MS/MS spectra of two ubiquitinated Miz1 peptides. (D) WT fibroblasts were transfected with expression vector encoding Xpress-Miz1 (WT or K388/473R mutant) (5 μg each), along with HA-Ub (5 μg) and then stimulated without or with TNF-α (5 ng/mL, 15 min). Ubiquitinated Xpress-Miz1 was analyzed as described in Fig. 3. (E) The effect of Xpress-Miz1 and Miz1(K388/472R) mutant on ubiquitination of cotransfected M2-TRAF2 was analyzed, as described previously (31). (F) WT and Miz1-null MEFs transfected with expression vector encoding M2-JNK1 (1 μg), along with or without Xpress-Miz1 (WT or K388/472R mutant) or empty vector (2 μg each) were treated without or with TNF-α (5 ng/mL, 15 min). M2-JNK1 activity and expression of M2-JNK1 and Xpress-Miz1 were determined.