Figure 2.

Insulin and 20E counteractively regulated the abundance and phosphorylation of INSRβ. In total, 7.5% SDS-PAGE gel for the experiments in addition to indication. A, insulin regulation in the expression of INSRβ in the larval epidermis (The purpose of using the epidermis in many places in this article is to keep it consistent with our HaEpi cells). Statistics were based on the ratio of the density of two bands to the ACTB band. The antibody against INSRβ was used. ACTB was used as the internal reference. B, insulin regulation on the phosphorylation of INSRβ after affinity enrichment of INSRβ. 12.5% gel. Antibodies against p-INSRβ and INSRβ were used, respectively. C, 20E regulation in the abundance of INSRβ in the larval epidermis. Statistics were based on the ratio of the density of two bands to the ACTB band. The antibody against INSRβ was used. ACTB was used as the internal reference. D, 20E regulation of the phosphorylation of INSRβ in the larval epidermis after enrichment of INSRβ. 12.5% gel. Antibodies against p-INSRβ and INSRβ were used, respectively. In total, 500 ng 20E was calculated as 5 μM 20E in vivo by the formula: c = m/MV, m = 500 ng, M = 480.63 g/mol, V ≈ 200 μl of a sixth-6 h larva. E, the effects of overlapped 20E and insulin on INSRβ levels and phosphorylation in HaEpi cells. 12.5% gel. Hormones were added 12 h after starvation (in Grace's medium without FBS). DMSO and PBS were used as solvent controls of 20E and insulin, respectively. The protein amount of each lane was 50 μg. Antibodies against p-INSRβ, INSRβ and ACTB were used, respectively. F, statistical analysis of (E) using ANOVA, different letters represented significant differences (p < 0.05). The statistical analyses of the related density of western blotting bands were performed using ImageJ software based on three independent replicates. The bars indicate the mean ± SD.