Fig. 3
Requirement of Kr-h1 phosphorylation in inhibiting E93 transcription. A Relative levels of E93 mRNA in mid-4th instar nymphs treated with dsKr-h1 vs. dsGFP control, NPC15437 (NPC) vs. DMSO solvent control (Cont.), and dsPKCα vs. dsGFP control. **P<0.01 and ***P<0.001. n=8. B Luciferase reporter assays using S2 cells co-transfected with pGL4.10-4×E93-623 to -606 plus pAc5.1/Flag-Kr-h1, pAc5.1/Flag-Kr-h1S154A, pAc5.1/Flag-Kr-h1S154D, or pAc5.1/Flag empty control with or without 10 μM methoprene treatment. Co-transfection of pGL4.10-4×E93-623 to -606 and pAc5.1/Flag empty control without methoprene treatment was used as the control. Means labeled with different letters indicate significant difference at P<0.05. n=4. C ChIP assays showing relative precipitation of E93 promoter region with the KBS motif (RPEP-KBS) in mid-4th (4M) and 5th (5M) instar nymphs. D RPEP-KBS in 4M nymphs treated with NPC15437 (NPC) vs. DMSO solvent control (Cont.) and dsPKCα vs. dsGFP control. E RPEP-KBS in 5M nymphs treated with 50 μg methoprene vs. acetone solvent control. In C–E, p-Kr-h1, phospho-Kr-h1 (Ser154) antibody; Kr-h1, Kr-h1 antibody; and IgG, non-specific rabbit IgG control. Means labeled with different letters indicate significant difference at P<0.05. n=4