Figure 5.

HERC3 mediates RelA K48 ubiquitination and protein destabilization. (A) His-ubiquitin, myc-RelA, with or without myc-HERC3 were transiently introduced into HEK293T cells. RelA was precipitated from whole cell lysates with RelA-specific antibody and the nature of RelA ubiquitination was examined by Western Blotting with anti-ubiquitin antibodies recognizing total, K48 or K63 ubiquitin, respectively. (B) HEK293T cells were transfected with myc-RelA with or without myc-HERC3. Twenty four hours after transfection, cells were pulse-labeled with [³⁵S]-methionine for 1 h and chased for 0, 2, 4 and 8 h. RelA was precipitated from total cell extracts with myc-affinity agarose, and protein levels were detected by autoradiography. (C) RelA was precipitated from transfected BAEC total cell extracts after pulse-labeling with [³⁵S]-methionine for 1 h and chasing for 0 and 8 h. Protein levels in precipitates were assessed by separation on SDS-PAGE and autoradiography. Average percent protein remaining before and after chase is indicated. All experiments were carried out at least in triplicates. h, hours; IB, immunoblot; IP, immunoprecipitation.