Gain-of-function conferred by RNF31 SNPs Gain-of-function conferred by RNF31 SNPs. A, control or RNF31 shRNAs were inducibly expressed in HBL1 cells that had been transduced with rescue vectors expressing RNF31 isoforms or with an empty vector. Gain-of-function conferred by RNF31 SNPs. A, control or RNF31 shRNAs were inducibly expressed in HBL1 cells that had been transduced with rescue vectors expressing RNF31 isoforms or with an empty vector. Doxycycline (Dox)-induced cells were lysed in 1% SDS, diluted, and then subjected to immunoprecipitation with an anti-NEMO antibody, followed by immunoblotting for indicated proteins (top). The relative NEMO linear ubiquitination signal intensity was determined by densitometric analysis (bottom). Also shown are immunoblots for the indicated proteins in whole-cell lysates from the same cells. B, HBL1 cells engineered to express indicated Myc epitope-tagged RNF31 isoforms were subject to anti-Myc immunoprecipitation followed by elution with Myc peptides. The elutions were examined in an E3 ligase activity ELISA assay (top) or by immunoblotting for the indicated proteins. C, protein prepared as in B were used in an in vitro ubiqutination assay, the products of which were analyzed by immunoblotting for the indicated proteins. D, anti-Myc immunoprecipitates prepared as in B or whole-cell lysates were analyzed by immunoblotting for the indicated proteins. E, densitometic quantititation of coimmunoprecipitation experiments demonstrating the association of RNF31 isoforms and RBCK as in D. All error bars, SEM of replicate experiments [n = 3 for all experiments except E (n = 11)]. Yibin Yang et al. Cancer Discovery 2014;4:480-493 ©2014 by American Association for Cancer Research