Fig. 4

Palmitate- and C6 ceramide-induced changes in Tnnt3 pre-mRNA alternative splicing are blocked by inhibition of PP2A. a-c L6 myotubes were pretreated for two hours with either 1 or 15 nM okadaic acid (OKA) or an equal volume of MeOH or DMSO for 24-h prior to Western blot analysis. The ratio of (a) GSK3β at S9 to total GSK3β, (b) ERK1/2 at T202/Y204 to total ERK1/2, and (c) phosphorylated Akt at S473 to total Akt was assessed. L6 myotubes were pretreated for two hours with okadaic acid or an equal volume of DMSO (Vehicle) prior to a 24-h treatment with (d) 150 μM PA conjugated to BSA (PA) or an equal volume of BSA alone or (e) 20 μM C6 ceramide (C6) or an equal volume of methanol (MeOH). The fold change in the relative abundance of the 737 base pair Tnnt3 splice form was assessed by capillary electrophoresis (d and e). Data are presented as means ± SEM from three independent experiments using three replicates per treatment. Statistical significance was assessed by Student’s t-test (a-c) or Two-way ANOVA with Fishers LSD post-hoc test for multiple comparisons (d and e). Statistically different means are denoted with an asterisk (*) or different letters above the bars (p ≤ 0.05). # p = 0.08 vs. BSA/Vehicle