Journal: Molecular and Cellular Biology
Article Title: Identification of the In Vivo Casein Kinase II Phosphorylation Site within the Homeodomain of the Cardiac Tisue-Specifying Homeobox Gene Product Csx/Nkx2.5
Figure Lengend Snippet: Phosphopeptide and phosphoamino acid analysis of Csx/Nkx2.5. (A) Tryptic phosphopeptide mapping of wild-type Csx/Nkx2.5 (a), CKII mutant (b), and in vitro CKII-phosphorylated MBP-homeodomain protein (c). Metabolically 32 P-labeled Csx/Nkx2.5 and the CKII mutant (Csx/Nkx2.5 163S-A ) were immunoprecipitated with anti-Csx/Nkx2.5 MAb (2D10), resolved by SDS-PAGE, transferred to a PVDF membrane, and autoradiographed. In vitro CKII-phosphorylated MBP-homeodomain protein was also electrophoresed and autoradiographed. These protein bands were cut out, digested with trypsin, and resolved on TLC plates by electrophoresis in the first dimension and chromatography in the second dimension. In vitro CKII-phosphorylated peptide (c [arrow]) which corresponded to peptide 1 in the wild type (a [arrow]), was markedly reduced in the CKII mutant (b). Arrowheads, sample loaded points; X, lysine marker. (B) Phosphoamino acid analysis of wild-type Csx/Nkx2.5 showed Csx/Nkx2.5 is phosphorylated predominantly in S and weakly in T.
Article Snippet: Immunoprecipitants of in vivo-labeled Csx/Nkx2.5 or in vitro-phosphorylated MBP-HD fusion protein were transferred to a polyvinylidene difluoride (PVDF) membrane (Millipore) after SDS-PAGE and then autoradiographed.
Techniques: Phosphoamino Acid Analysis, Mutagenesis, In Vitro, Metabolic Labelling, Labeling, Immunoprecipitation, SDS Page, Thin Layer Chromatography, Electrophoresis, Chromatography, Marker