Fig. 3
RNF5 catalyzes the formation of K-48 type ubiquitin chains on K63 site of E. a E was ubiquitinated via K48-linked but not K6, K11, K27, K29, K33, or K63. b knockdown of RNF5 decreased K48-linked ubiquitination of E. Lysates of RNF5-silenced HEK293T cells transfected to express E-HA and ubiquitin-K48-Flag were subjected to HA IP, and then analyzed by IB. c RNF5 ubiquitinates E on K63. HEK293T cells transfected to express ubiquitin-K48-Flag and E-HA, EK53R, EK63R, or EKK53,63RR, cell lysates were subjected to Flag IP and the proteins of interest in the precipitates were detected by IB. d K63 is the site being ubiquitinated. Standard dropout reactions containing the indicated components were allowed to proceed for 4 h and the proteins were resolved by SDS-PAGE and detected by Coomassie blue staining. e The EK63R mutant was recalcitrant to RNF5-mediated degradation. HEK293T cells transfected to co-express RNF5-Flag with E-HA or its K53R or K63R mutants were lysed and the level of relevant proteins was detected by IB
