Hamster cheek pouches were treated with buffer (Control) or 10 μm or 100 μm ACh, during a period of 1.5, 3 or 6 min, and then homogenised and prepared for Western blot analysis of the microsomal fraction. Top, time course of ACh-induced microsomal eNOS translocation. The boxes show Western blots of representative pools of each experimental group (80 μg total protein per lane). Pools were prepared using equal-protein aliquots of every individual tissue in each group. The underlying bar graph shows the corresponding densitometric image analysis of band intensities of individual hamsters (mean ± s.e.m.). Bottom, the same pools and individual tissue samples were analysed by Western blot and densitometric analysis for Cav-1. The number of animals in each group is shown inside the bars in the lower panel. Western blots were revealed with diaminobenzidine. * P < 0.05vs. control; † P < 0.01vs. control and vs. 10 μm ACh (ANOVA, Newman-Keuls test).