Supporting information for Kurokawa et al. (2003) Proc. Natl. Acad. Sci. USA, 10.1073/pnas.0434935100
Fig. 5.
PKA phosphorylation of KCNQ1 does not require the presence of KCNE1. Chinese hamster ovary cells were incubated with or without cAMP/okadaic acid (OA) (0.3 mM and 0.1 µM, respectively) at 37ºC for 15 min. Cell lysates were prepared as described in Materials and Methods. An anti-KCNQ1 antibody was used to immunoprecipitate KCNQ1 channel proteins. Immunoprecipitates then were incubated with protein kinase A (PKA) and ATP, which contained 10% [γ-32P]ATP, at room temperature for 5 min. After size-fractionizing, phosphorylated KCNQ1 channels were detected by autoradiography. (Upper) Back-phosphorylated KCNQ1 channels (upper bands) with or without prior cAMP/OA incubation. cAMP/OA incubation phosphorylated KCNQ1. Thus, less KCNQ1 can be phosphorylated further by PKA. The back-phosphorylation assay also indicates that phosphorylation of KCNQ1 does not depend on KCNE1. (Lower) KCNQ1 immunoblots from the immunoprecipitates, verifying that comparable amount of KCNQ1 channel proteins were subject to the back-phosphorylation assay. The lower bands in both Upper and Lower are IgG proteins.