FIG 5.

MC132 expression causes a depletion of p65 protein expression. (A) Effect of inducible MC132 expression on TNF-α-stimulated NF-κB activation. HEK293T cells stably transfected with pMEP4 and pMEP4-MC132 were seeded at 6 × 10⁵ cells per well in 6-well dishes and treated with (+) or without (−) 1 μM CdCl₂ to induce MCV protein expression. Twenty-four hours later, cells were stimulated with 50 ng/ml TNF-α for the indicated times, and cell lysates were immunoblotted with the indicated antibodies. Representative blots are shown (n = 3). (B) HEK293T cells stably containing pMEP4-MC132 or control cells containing pMEP4 were seeded at 6 × 10⁵ cells per well in 6-well dishes for 24 h with the indicated concentrations of CdCl₂ to induce MC132 expression, and cell lysates were immunoblotted for p65 and MC132. Representative blots are shown (n = 3). (C) HEK293T cells were seeded at 6 × 10⁵ cells per well in 6-well dishes, transfected with 3 μg pCEP4-MC132, fixed 24 h later, and stained with DAPI (blue), for MC132-Flag (green) and endogenous p65 (red), and with an anti-rabbit isotype control. Representative images are shown (n = 3). MC132-positive cells are indicated with white arrows. (D) HEK293T cells were seeded at 2 × 10⁵ cells per ml and transfected with the indicated amounts of p65 expression vector, with or without 50 ng of pCEP4-MC132. Cells were harvested 24 h later and assayed for NF-κB reporter gene activity. Data are presented as percentages of reporter activation by increasing amounts of p65 and display the effect of 50 ng of MC132 on this activation, with data given as means ± SD for triplicate samples from a representative experiment (n = 3). *, P < 0.001 compared to the control.