Fig. 2
From: The integrated stress response regulates BMP signalling through effects on translation
dPPP1R15 or dGCN2 affects MAD phosphorylation in the developing wing. a Representative fluorescence micrographs of pupal wings of the indicated genotypes at 30 h after pupariation stained red for pMAD. Open arrowheads indicate ACV territory. Closed arrowheads indicate ectopic pMAD signal. Scale bars = 100 μm. b Representative fluorescence micrograph of pupal wings of the indicated genotypes at 30 h after pupariation. Green fluorescence indicates activation of the dad-GFP.N reporter. Scale bars = 100 μm. c Immunoblot of cell lysates: lanes 1–4, S2 cells stably transfected with V5.pMT-Puro; lanes 5–8, S2 cells stably transfected with dGCN2-CA-V5.pMT-Puro. Cu2+ indicates treatment with 0.7 mM copper sulphate for 16 h; dpp indicates treatment with 1 nM Dpp for 1 h prior to lysis. dGCN2-CA-V5 was detected with anti-V5 antibody. crc, pMAD and actin were detected using specific antibodies. d Quantification of pMAD staining in c with strongest signal with each experiment set as 1. n = 3. P value calculated using analysis of variance (ANOVA) with Bonferroni post hoc testing. e S2 cell lysates: lanes 1–3, V5.pMT-Puro S2 cells; lanes 4–6, dGCN2-CA-V5.pMT-Puro S2 cells. Cu2+ indicates treatment with 0.7 mM copper sulphate for the indicated times. 35S-labelled cysteine and methionine were added to cells for 10 min prior to lysis. 35S-labelling indicates autoradiograph. Coomassie staining served as a loading control. f Immunoblot of S2 cell lysates expressing FLAG-MAD. CHX indicates treatment with 14 μg/ml cycloheximide for the indicated times. dpp indicates treatment with 0.5 nM dpp for 1 h prior to lysis. FLAG-MAD was detected with an anti-FLAG antibody. pMAD and actin were detected with specific antibodies. Filled arrowhead indicates phosphorylated MAD-FLAG; open arrowhead indicates endogenous pMAD. g Quantification of phosphorylated FLAG-MAD (pMAD) and (h) total FLAG-MAD from f, both normalised to actin signal with the strongest signal in each experiment set as 1. n = 3. P value calculated using ANOVA with Bonferroni post hoc testing