Co-immunoprecipitation of hTTP with potential interacting partners.

<p>In this and subsequent figures, extracts were prepared in RIPA buffer from HEK 293 cells transfected with DNA encoding the HA- and Flag-tagged expression vectors indicated at the top of each gel lane by the “+” sign. Total DNA transfected was 5.0 µg per 100 mm petri dish. For each western blot shown, the immunoprecipitating antibody (IP) and the subsequent immunoblotting antibody (IB) are indicated to the left of each panel, as are the positions of protein molecular weight standards. The immunoreactive protein species are indicated by the labeled arrows to the right of each blot. Each immunoprecipitation used 1 mg of cellular lysate protein as the starting material. In some cases, the blots are of whole cell lysates (WCL) (50 µg of total protein per lane) instead of from immunoprecipitations to confirm expression of the respective protein in the lysates prior to immunoprecipitation. In addition to the epitope-tag antibodies indicated, western blotting in this case also used antibodies to endogenous nucleolin (NCL), CIN85, and HSP70. See the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0009588#s2" target="_blank">Results</a> section for additional details.