Fig. 3

crc regulates wing venation and antagonises MAD phosphorylation. a Representative photomicrographs (5× objective) of adult wings of the indicated genotypes. En indicates enGAL4 driver control. en > crc RNAi indicates enGAL4 > UAS-crc RNAi. en > ppp1r15 RNAi indicates enGAL4 > UAS-ppp1r15 RNAi. en > ppp1r15 RNAi;crc RNAi indicates enGAL4 > UAS-crc RNAi;UAS-ppp1r15 RNAi. Lower panels are enlargements of the crossvein territories. Scale bars = 250 μm. b Quantification of ACV phenotype in a. P values calculated using X² statistics with Bonferroni correction for multiple comparisons. c In situ hybridisation of w¹¹¹⁸ pupal wings with sense or antisense probes to residues 1405–1900 of crc transcript A. Scale bars = 250 μm. d Representative fluorescence micrograph (40× objective) of wing imaginal discs: signal = pMAD. En indicates enGAL4 driver control. en > crc indicates enGAL4 > UAS-HA-crcA. Orientation: left = anterior. Arrowhead indicates expected position of posterior pMAD zone. Scale bars = 50 μm. e Representative photomicrographs of adult wings of the indicated genotypes. En indicates enGAL4 driver control. en > crc indicates enGAL4 > UAS-crc. Scale bars = 250 μm. f Immunoblot of S2 cell lysates: lanes 1–4, S2 cells stably transfected with HA.pMT-Puro; lanes 5–8, S2 cells stably transfected with HA-crcA.pMT-Puro. Cu²⁺ indicates treatment with 0.7 mM copper sulphate for 24 h. dpp indicates treatment with 0.5 nM dpp for 1 h prior to lysis. HA-crc was detected with anti-HA antibody. pMAD and actin were detected using specific antibodies. g Quantification of pMAD staining in f with highest signal per experiment set as 1. n = 5. P value calculated using analysis of variance (ANOVA) with Bonferroni post hoc testing