Fig. 13.

Time Course of UCN-II-Stimulated ERK1/2 (A) and p38MAPK (B) Phosphorylation in HEK293 Cells Transiently Expressing Wild-Type or Mutant CRH-R2β

HEK293 cells transiently expressing wild-type or mutant CRH-R2β receptors were treated with or without UCN-II (100 nm) for various time intervals to induce MAPK activation. After cell lysis and centrifugation, supernatants were subjected to SDS-PAGE and immunoblotted with antibodies for phospho-ERK1/2 and total-ERK1/2 to determine the phosphorylated/activated ERK1/2 and secondary antibodies conjugated to IRDye 800 and Alexa Fluor 680 (near-IR fluorophore dyes) as described in Materials and Methods. Alternatively, samples were immunoblotted with antibodies for phospho-p38MAPK and total p38MAPK. The data represent the mean ± sem of three estimations from three independent experiments.