Fig. 3.

Characterization of FXYD1 phosphospecific antibodies. A: the reactivity of CP63, CP68, CP69, and CP689 to in vitro phosphorylated recombinant FXYD1 and its point mutants was investigated by dot blotting in vitro phosphorylation reactions that had proceeded to completion. Antibody CP69 was preincubated with 10 μg/ml unphosphorylated blocking peptide, and antibody CP689 was preincubated with unphosphorylated blocking peptide plus two singly phosphorylated blocking peptides (all 10 μg/ml), as described in materials and methods. All phosphospecific antibodies show the appropriate reactivity, although note that mutation T69A severely limits the ability of antibody CP68 to bind to FXYD1 phosphorylated at serine 68. Antibody CP689 shows very weak binding to mutant S68A phosphorylated by PKC (i.e., phosphorylated at T69 only); however, the majority of signal generated by this antibody is for dual phosphorylated S68 and T69 [compare with signal from wild type (WT) and S63A]. B: time course of phosphorylation of wild-type recombinant FXYD1 by PKA and PKC. Phosphorylation of S63 by PKA is very slow compared with S68 (phosphorylation of S68 is complete within 10 min, but phosphorylation of S63 requires 90 min). Phosphorylation of T69 by PKCɛ severely limits binding of antibody CP68 to phosphorylated S68.