Figure 5
From: Capping Enzyme mRNA-cap/RNGTT Regulates Hedgehog Pathway Activity by Antagonizing Protein Kinase A
mRNA-cap regulates PKA activity. (A,B) The PKA activity (A) and cAMP level (B) were measured from control wing discs or wing discs expressing mRNA-cap RNAi with MS1096. Approximately 400 discs were dissected and lysed for each assay. (C–C”’) Overexpression of HA-PKA-C2 by MS1096 was immunostained for Ci, ptc-lacZ and HA. (D–D”’) Overexpression of HA-PKA-C3 by MS1096 was immunostained for Ci, ptc-lacZ and HA. The levels of Ci were decreased. (E–F”’) Overexpression of HA-PKA-R1 (E–E”’) or Myc-PKA-R2 (F–F”’) with MS1096 was immunostained for Ci, ptc-lacZ and HA or Myc. The levels of Ci and ptc-lacZ were up-regulated. (G) The relative mRNA levels of PKA-C1, PKA-C3, PKA-R1 and PKA-R2 of wing discs were measured by RT-qPCR upon knockdown of mRNA-cap. MS1096 acted as a control. (H) Western blot analysis of lysates from control wing discs or wing discs expressing mRNA-cap RNAi with MS1096. Approximately 100 discs were dissected, lysed, and blotted with anti-PKA-C1 antibody, respectively. (I) Western blot analysis of PKA-R1 levels from control wing discs or wing discs expressing mRNA-cap RNAi. Approximately 100 discs were used. (J,K) Knockdown of mRNA-cap did not affect the dimer formation of PKA-R1 (J) or PKA-C1 (K). (L,M) mRNA-cap didn’t change the interaction between PKA-C1 and PKA-R1. All images are representative of three independent experiments. (A,B,G,H and I) Data presented are the average of three independent experiments and error bars represent SD. ***P < 0.001, t-test.
