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. 2016 Jul 26;2:16019. doi: 10.1038/celldisc.2016.19

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Copyright © 2016 SIBS, CAS

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RNF126 is an E3 ubiquitin (Ub) ligase for PDKs. (a) Western blot analysis of the ubiquitination of PDK1-FLAG and LDHA-FLAG by V5-RNF126 in HEK293 cells. (b) Western blot analysis of FLAG immunoprecipitates (IPs) indicating that RNF126 interacts with PDK1, but not with LDHA, in HEK293 cells. (c) Western blot analysis of PDK1-FLAG ubiquitination demonstrating that the RING domain of RNF126 is necessary for PDK1 ubiquitination. (d) Forced expression of shRNF126-resistant RNF126 decreased PDK1 protein in attached PC8 cells that were depleted of endogenous RNF126. (e) Forced expression of shRNF126-resistant RNF126 decreased lactate production in attached cells depleted of endogenous RNF126. Error bars indicate the s.d. (n=3). The data were analyzed using a t-test. **P<0.01. (f) Amino-acid sequence alignment of the PDK family. (g) Western blot analysis showing ubiquitination of wild-type PDK1-FLAG or K/R mutant PDK1-FLAG. The data shown in (a–e and g) are representative of three independent experiments with similar results. HA, hemagglutinin; IB, immunoblotting; WCL, whole-cell lysate.