Ubiquitination is a key regulatory mechanism in the immune deficiency (IMD) pathway in Drosophila. In this study, we first developed a simple immunoblot method to identify components involved in this pathway. Considering the emerging roles of ubiquitin-conjugating enzymes (E2s) in determining ubiquitin chain types and ubiquitination speed, we screened for E2s required for IMD activation. We found that UbcD4, in addition to the previously reported E2s Effete and Bendless, was required for activation of the IMD pathway. RNAi-mediated knockdown of the UbcD4 ortholog, E2-25K/Ube2K, inhibited TNFccand LPS-mediated activation of the NF-KB pathway, implying that UbcD4 and E2-25K/Ube2K play a conserved role as positive regulators in both pathways. (C) 2015 Elsevier Inc. All rights reserved.