FIG. 7.

Endogenous RMP associates with RNA polymerase II complex. (A) Western blot analysis of molar ratio of endogenous RMP to RPB5 in HepG2 cells. Proteins were loaded in lanes as follows: lanes 4 to 6, 60, 40, and 20 μg, respectively, of HepG2 total cell lysate; lanes 1 to 3, 15, 5, and 2.5 ng, respectively, of bacterially expressed recombinant FLAG-RMP; and lanes 7 to 9, 2, 10, and 5 ng, respectively, of FLAG-RPB5. Proteins were fractionated by SDS-PAGE and subjected to Western blot analysis with anti-RMP (upper panel) and anti-RPB5 (lower panel) antibodies. (B) Coimmunoprecipitation of RMP with RNA polymerase II subunits. HepG2 cell lysates were precipitated with the indicated antibodies. The immunoprecipitates (IP) together with an aliquot of the lysate (Input 5%), were separated by SDS-PAGE and detected with antibodies directed against RPB1 (anti-CTD), RMP (α-RMP), RPB5, and RPB6 (α-RPB6). The asterisk shows the heavy chain of the first antibody used in the immunoprecipitation. α-Tub, antitubulin.