Muscle-Type Specific Autophosphorylation of CaMKII Isoforms after Paced Contractions
Figure 2
Calcium/calmodulin-dependent kinase II isoforms in rat skeletal muscle. Total homogenate of a gastrocnemius muscle from a rat was prepared and subjected to in vitro kinase reactions in the presence of EGTA or calcium/calmodulin and was subjected to immunoblotting with a pan-CaMKII (a) and phospho-Thr287 specific antibody (b). CaMKII isoforms were then assigned based on a calcium/calmodulin-inducible phospho-Thr287 signal and a detection of similar sized bands according to the nomenclature established by Rose [12] and Bayer [13]. (c)(d) CaMKII isoforms (c) and Thr287 phosphorylated CaMKII (d) in equal protein amount in total homogenate of fatigue resistant (i.e., oxidative) and fatigable (i.e., glycolytic) compartments of the fast gastrocnemius medialis muscle and the slow fatigue resistant soleus muscle. Lanes originating from different parts of SDS-PAGE gels are separated by black lines. A second band at the height of the βM isoform is detected in soleus muscle. (e) Graph displays COXIV levels in the oxidative and glycolytic compartments of gastrocnemius medialis as determined by western blotting followed by immunodetection. Bars represent mean COXIV levels and symbols represent the levels in individual sample pairs. Symbols being connected by a stippled line reflect intra-animal pairs. The P-level of the difference between red and white GM is indicated (paired -test). (f) Example immunoblot showing the detection of COXIV protein and the actin loading control in the oxidative and glycolytic compartments of gastrocnemius medialis.
