(a, b) TRAF1 expression induces the ubiquitination of ERKS. HA-ERKS was co-expressed with TRAF1-VS along with Flag-ubiquitin in HEK293T cells. Cells were harvested, and then samples were prepared for Western blot analysis. HA-ERKS or Flag-ubiquitin was immunoprecipitated with anti-HA or anti-Flag and then ubiquitinated-ERKS was detected by immunoblot analysis. (c) TRAF1 expression induces the ubiquitination of ERKS on K184. HA-ERKS WT or the K171 R, K184R, or K349R mutant protein was co-expressed with TRAF1-VS, along with Flag-ubiquitin in HEK293T cells. HA-ERKS was immunoprecipitated with anti-HA, and then ubiquitinated-ERKS was detected by immunoblot analysis. ERK, extracellular signal-regulated kinase; HA, hemagglutinin; HEK, human embryonic kidney; IP, immunoprecipitation; K, keratin; KR, lysine to arginine; Ub, ubiquitin; WCL, whole cell lysates; WT, wild type.