Brap2 attenuates TNF-α-induced NF-κB translocation to nucleus.

<p>(A) HeLa cells were transfected with either Flag-Brap2 WT or Flag-Brap2 CA and stimulated with or without 5 ng/ml TNF-α for 30 min, and were subjected to immunocytochemistry using anti-Flag or anti-RelA/p65 antibodies. Cells that express certain amount of Flag-Brap2 are marked by dot lines and control cells are marked by solid line. RelA/p65 translocates in nucleus after the stimulation. The right panels show the same cells using a rainbow color. Bars, 50 µm. (<b>B</b>) Ratiometric measurement of RelA/p65 fluorescence observed in cells expressing Flag-Brap2 before and after treatment with 5 ng/ml TNF-α (n = 50; mean ± SEM; *** p<0.0001 by one-way ANOVA). (<b>C</b>) HEK293T cells were transfected with HA-Cul1 and Flag-Brap2 WT or CA, and stimulated with or without 5 ng/ml TNF-α for the indicated time, and subjected to immunoprecipitation (IP) with Flag antibody. The total cell lysates and IP were immunoblotted (IB) with indicated antibodies. (<b>D</b>) HEK293 cells were stimulated with 5 ng/ml TNF-α for indicated time, and cell lysates were subjected to immunoblot analysis.