FIG. 2.

Analysis of DMPK isoforms and mutants by Western blotting. Individual mouse DMPK cDNA products were analyzed by Western blotting using an anti-DMPK antibody. (A) Expression of isoforms DMPK A to F. The presence or absence of the VSGGG motif, the type of C-terminal tail, and the theoretical molecular weights are summarized below the lanes. The apparent molecular weights of the main bands are indicated (arrows). All isoforms with a VSGGG motif showed two distinct bands (arrowheads), whereas isoforms without a VSGGG motif showed only one (sometimes broad) band. (B) Expression of His-DMPK A through F kd mutants. All kd isoforms showed only one band (i.e., the lower band in A). (C) The effect of in vivo treatment with the phosphatase inhibitor OA (1 μM, 1 h) was tested for DMPK E, His-DMPK E kd, and DMPK E^VA. OA treatment changed the doublet ratios of DMPK E and E^VA but did not change the migration of His-DMPK E kd. Note that due to the His tag (2.4 kDa) DMPK E kd migrated slightly more slowly than the regular DMPK E isoform. Shown are results obtained by immunoprecipitation. (D) Ser379 mutations alter DMPK E mobility. DMPK E and mutants DMPK E^VA and E^VD were expressed and treated with OA prior to lysis as in panel C. Lysis was done at −80°C in 10% TCA-10 mM DTT-acetone to preserve intracellular phosphorylation status.