Fig. 4.

XIAP enhances the association of Elongin B/C with SOCS1 and promotes SOCS1 K63 ubiquitination. a XIAP increases the association of Elongin B/C with SOCS1. Full-length, ΔRING (ΔR) or C-terminal (C) XIAP-FLAG was co-transfected with SOCS1-HA and Elongin B/C-Myc into HEK293T cells. Elongin B/C-Myc in cell lysates was pulled down by anti-Myc and the presence of SOCS1 and XIAP in the precipitates and lysates was determined by the respective antibodies. b XIAP enhances the addition of K63 ubiquitin to SOCS1 in vivo. XIAP-FLAG was co-transfected with SOCS1-myc and Elongin B/C-Myc into HEK293T cells, with WT Ub, K48 Ub or K63 Ub, as indicated. SOCS1 was immune-precipitated, and the association of ubiquitin type determined. c XIAP promotes SOCS1 K63 poly-ubiquitination in vitro. Recombinant K63 ubiquitin-HA, E1, E2 (UBC13), SOCS1-HA (on Mag beads), Elongin B/C-Myc, or XIAP-FLAG was added as indicated to a ubiquitination reaction conducted at 30 °C for 1 h. Around 10% of the reaction mixture was used for Western blotting. SOCS1-HA was pulled down from the rest of the reaction mixture and ubiquitination of the SOCS1 complex was determined. Experiments were independently repeated three times (a, c) or twice (b)