Fig. 5. Effects of 20-hydroxyecdysone (20E) injection on expression of guanylyl cyclases in hepatopancreas, testis, claw muscle and thoracic muscle. Intact land crabs were injected with 20E or vehicle (10% ethanol) and tissues collected at 4 h, 8 h and 12 h after injection. Total RNA was DNase-treated, reverse-transcribed and PCR-amplified using sequence-specific primers (see Materials and methods). PCR products after 35 cycles were resolved on 2% agarose gels (for Gl-GC-Iß, Gl-GC-III and Gl-EF2) or 10% polyacrylamide gels (for Gl-GC-II), stained with Ethidium Bromide, and quantified by scanning densitometry. GC expression was normalized to Gl-EF2 (values are mean ± 1 s.e.m., N=3). Significant differences between means, obtained by one-way ANOVA post-hoc test (Bonferroni–Dunn), are indicated with brackets (a, P<0.05; b, P<0.01; c, P<0.002).