Figure 3. Generation of muscle-specific SNARK transgenic mice.
(A) Transgenic mice with muscle-specific overexpression of FLAG-tagged wild-type SNARK (SWT) or a dominant-negative inactive SNARK mutant (T208A; SDN) were generated using the MCK promoter. (B) Phosphotransferase activity of SWT and SDN constructs against the AMARA peptide was assessed by immunoprecipitation of FLAG from 293 HEK overexpression lysates using 32P-γ-ATP. n = 5 per group. (C) The dominant-negative nature of the SNARK dominant-negative construct was verified by measuring endogenous SNARK phosphorylation at its activating site T208 in 293 HEK cells transfected with PCDNA3.1 empty vector (EV), SNARK dominant-negative mutant (SDN), or wild-type human SNARK (SWT). n = 4 per group. (D) A FLAG immunoblot demonstrating the expression of SNARK transgene in the TA, extensor digitorum longus (EDL), soleus (SOL), and heart muscles compared with wild-type littermates, and the absence of expression in livers of SNARK transgenic mice. AMPK is shown as a loading control. (E) Relative expression levels of transgene and endogenous protein in the muscles of SNARK transgenic mice and littermate controls using FLAG, phospho-SNARK T208, and total SNARK antibodies. Two lines of transgenic mice overexpressing different levels of dominant-negative SNARK (SDN-Low and SDN-High) were studied, and data from two independent lines of SWT mice with similar expression levels were pooled for analysis. *P < 0.05 vs. control determined by 1-way ANOVA and Bonferroni post-hoc testing. Error bars indicate mean ± SEM.