FIG 7.

MC132 recruits p65 to a Cullin-5/Elongin B/Elongin C complex to target p65 for ubiquitin-mediated degradation. (A) Volcano plot of MC132-interacting proteins identified from MC132-expressing HEK293T cells by AP-MS. (B) MC132 targets p65 to the Elongin B/C complex. HEK293T cells were seeded at 4 × 10⁶ cells per 10-cm plate and transfected with 2 μg expression vector(s) for MC132-HA, Elongin B-Flag, and/or Elongin c-Myc, as indicated. Twenty-four hours later, cells were lysed, immunoprecipitated with anti-Flag beads, and then immunoblotted with the indicated antibodies. Representative blots are shown (n = 3). (C) MC132 induces polyubiquitination of p65. HEK293T cells were seeded at 4 × 10⁶ cells per 10-cm plate, transfected with 4 μg expression vector(s) for ubiquitin-HA, MC132-Flag, and/or T3-Flag, as indicated, and treated with 20 μM MG132 for 4 h prior to harvest. Twenty-four hours later, cells were lysed, immunoprecipitated with anti-Flag beads, and then immunoblotted with the indicated antibodies. Ubi-p65, polyubiquitinated p65. A representative blot is shown (n = 3). (D) HEK293T cells were seeded at 2 × 10⁵ cells per ml in 24-well dishes and transfected with 2.5 nM Elongin B siRNA at 24 and 48 h, and then cDNAs from cells were assayed for levels of Elongin B normalized to β-actin by real-time PCR. (E) HEK293T cells were seeded at 4 × 10⁶ cells per 10-cm plate, transfected with 2.5 nM Elongin B or control siRNA for 24 h and again at 48 h, and transfected with the indicated constructs prior to MG132 treatment for 4 h and harvest. Total p65 was immunoprecipitated, and immunoblots were probed with the indicated antibodies. (F) The effect of Elongin B siRNA on TRAF6-stimulated NF-κB activation and MC132 inhibition was measured by a reporter gene assay as described in the legend to Fig. 1. Data are percentages of the stimulation activity for control cells and are means ± SD for triplicate samples from a representative experiment (n = 4). *, P < 0.001 compared to the control. (G) HEK293T cells were seeded and treated as described for panel E, but they were transfected with 25 μM Cullin-5 or control siRNA for 24 h and then immunoblotted for Cullin-5 expression. (H) Cullin-5 siRNA reduces MC132-stimulated p65 ubiquitination. Cells were treated as described for panel C, except that Cullin-5 or control siRNA was transfected prior to MG132 treatment and harvest. Representative blots are shown (n = 4).