Fig. 2

Insulin promotes PGK1 phosphorylation and represses PGK1 acetylation, but 20E does the opposite in vivo. A Vmax of 3-PG production in the fat body and 20E titers (according to [25]) from 5F to adult stages. p < 0.05, n = 3. B Levels of PGK1, PGK1 phosphorylation, and PGK1 acetylation in the fat body from 5F to adult stages. PGK1 proteins from the fat body were purified with CNBr-activated Sepharose 4B and an anti-PGK1 monoclonal antibody, followed by in vitro detection. 5F, fifth-instar feeding larvae; 5 M, fifth-instar molting larvae; 6th-6 h to 6th-120 h represent sixth-instar larvae at the corresponding hours. P-0 d to P-8 d denote 0- to 8-day-old pupae. F, feeding; M, molting; MM, metamorphic molting; P, pupae. An additional band at about 40 kDa appeared in pY immunoprecipitation indicated as Unknown. n = 3. C Insulin increased PGK1 phosphorylation but decreased its acetylation. Sixth-instar larvae at 48 h were injected with 5 μg insulin/larva for 3 h. *p < 0.05, n = 3. D Insulin enhanced PGK1 glycolytic activity based on the detection of Vmax for 3-PG production in C. *p < 0.05, n = 3. E 20E reduced PGK1 phosphorylation but increased its acetylation. Sixth-instar larvae at 48 h were injected with 500 ng 20E/larva for 3 h. *p < 0.05, **p < 0.01, n = 3. F As measured by the Vmax for 3-PG production, 20E decreased PGK1 glycolytic activity in E. *p < 0.05, n = 3. PGK1 proteins from the fat body were purified by CNBr-activated Sepharose 4B with anti-PGK1 monoclonal antibody before western blot analysis and the detection of Vmax for 3-PG production. 12.5% SDS-PAGE gels were used for western blot and β-actin was used for protein quantity control. Data represent means ± SD for three independent experiments with five larvae per replicate. Statistically significant differences were calculated using two-tailed Student’s t tests (*: p < 0.05, **p < 0.01) or one-way analysis of variance (ANOVA, p < 0.05) tests. pY, phosphorylated tyrosine. Ac, acetylated lysine