Figure 5.

Insulin and muscle contractions do not affect accessibility of GLUT4 NH₂- or COOH-terminal epitopes. Teased single fibers, each ∼9-mm long, were prepared from basal (Bas), insulin- (Ins), contraction- (Ex) and insulin- and contraction-stimulated muscles (ExI). They were permeabilized and stained with antibodies to the COOH- or NH₂-terminal part of GLUT4 or to caveolin-3. Primary antibody binding was quantitated by measuring the amount of ¹²⁵I-labeled secondary antibody binding. Amount of bound GLUT4 antibodies is expressed as percent of caveolin-3 binding to fibers from the same muscle in order to correct for possible variations in sarcomere length during fixation. Results are presented as mean ± SEM of two or three separate experiments with 10–15 fibers per experiment. Analysis of variance (ANOVA) did not detect any significant difference between stimulation states within each antibody group (P > 0.05). Omission of the primary antibodies resulted in less than 2% of the labeling obtained with primary antibodies against GLUT4.