Figure 1.

Western blotting showing the decrease in INSRβ levels and phosphorylation during metamorphosis. A, the expression levels of INSRβ in tissues detected by antibody against INSRβ. 5F, fifth instar feeding larvae; 5M, fifth instar molting larvae; sixth-6 h–sixth-120 h, time stages of sixth instar larvae; P 0–P 8, 0-day-old to 8-day-old pupae; Adult, adult; F, feeding; M, molting; MM, metamorphic molting; P, pupae stage. ACTB was detected by the monoclonal antibody ACTB Rabbit mAb as the internal control. The loading quantity for each lane is 50 μg proteins. 12.5% SDS-PAGE gel. B, quantitative analysis of the INSRβ expression using ImageJ software. C, variation of INSRβ phosphorylation levels in epidermis during larval development detected by antibody against p-INSRβ. P-INSRβ was the phosphorylated form of INSRβ. The amount of INSRβ was affinity-enriched using the antibody as the method described. Quantitative analysis of the INSRβ phosphorylation using ImageJ software. Error bar was based three biological replicates; 12.5% SDS-PAGE gel. D, the variation of expression and phosphorylation levels of INSRβ in the epidermis from sixth instar 24 h larvae to 4-day-old pupae. The antibody against INSRβ was used. ACTB was used as the internal reference; 7.5% SDS-PAGE gel. Statistics were based on the ratio of the density of two bands to the ACTB band using ImageJ software. Statistical analysis was conducted using ANOVA, different letters represented significant differences (p < 0.05). The bars indicate the mean ± standard deviations (SD) of three times repetition.