Fig. 4.

JNK is the upstream kinase for SMAD2 linker phosphorylation in muscle. a–c EDL and TA muscles from wild-type control (WT; MCK-Cre^−/+) and muscle-specific JNK1/2 knockout mice (KO) were stimulated via in vitro (EDL) and in situ (TA) contraction. Immunoblotting of phosphorylated and total SMAD2 and JNK was performed (a). SMAD2-linker (SMAD2L) phosphorylation in response to in vitro contraction (b) and in situ contraction (c) was blunted in muscle JNK knockout mice. Each data point represents the basal and contraction results from an individual animal joined by a line. *P < 0.05, ***P < 0.01 vs Basal from the same genotype by repeated measures two-way ANOVA and Sidak’s post hoc testing. Main effects of genotype are displayed with P-values. d C2C12 myoblasts were treated with the JNK activator anisomycin (5 μM) for 30 min. e C2C12 myoblasts were transfected with plasmids expressing constitutively active JNK1, JNK2, or a combination of JNK1 and 2. Empty vector (EV) transfected cells were used as a control. f The ability of JNK to directly phosphorylate SMAD2 was assessed using an in vitro kinase assay. C2C12 lysates expressing empty vector (EV), or constitutively active JNK1 or JNK2 were purified by FLAG immunoprecipitation and incubated with recombinant SMAD2 and SMAD3 proteins. For tissue culture experiments (d–f), three independent experiments were performed and the data from one representative experiment, including all replicates is displayed. In all experiments, JNK activation (pJNK) and SMAD2 linker phosphorylation (pSMAD2L) were assessed by immunoblotting. Images obtained using stain-free gel technology (Bio-Rad) that allows for total protein visualization and quantification are shown as a loading control (Loading). pJNK phosphorylated (active) C-Jun N-terminal Kinase, pSMAD2L, linker-region phosphorylated SMAD2; SMAD2, total SMAD2; pP38, phosphorylation P38 MAPK; pERK, phosphorylated extracellular signal regulated kinase