Supplementary Figure 4: Transgenic HRG-7 rescues hrg-7 mutant phenotypes.
From: Inter-organ signalling by HRG-7 promotes systemic haem homeostasis
(a) Cartoon depicting the deleted region of hrg-7 in the tm6801 allele. Deletion is depicted by an underline. Open rectangles, exons; gray boxes, untranslated regions. (b) Homology model of HRG-7 using I-TASSER. Left: the putative active site is indicated by a red circle. The conserved disulfide bonds are indicated by white arrows. Alpha helixes are colored fuchsia. Beta sheets are colored yellow. Right: Zoomed in image of the putative active site. Yellow arrows indicate the catalytic aspartic acid residues and the conserved tyrosine of the flap region. (c) Immunoblot analysis of HRG-7 expression in worms expressing WT HRG-7 or HRG-7TM6801 and fed dsRNA against control vector or hrg-7. Membranes were probed with polyclonal anti-HRG-7 antibody and then incubated with HRP-conjugated anti-rabbit secondary antibody. * indicates pro-HRG-7. + indicates mature HRG-7. “Low” indicates low sensitivity detection. “High” indicates high sensitivity detection. Unprocessed blots are shown in Figure S6. Data is representative of three independent experiments. (d) GFP fluorescence quantified from IQ6011 expressing WT hrg-7, hrg-7(tm6801), or hrg-7(tm6801) and transgenic Pvha−6::hrg-7PR grown on OP50. GFP is presented as fold change compared to wild type (WT) worms. ∗∗∗P < 0.001, ∗∗P < 0.01, ∗P < 0.05 (one-way ANOVA). Graph represents the mean and SEM of three biological independent experiments. N = 120 worms per treatment per experiment. See Supplementary Table 3 for statistics source data. (e) Immunoblot analysis of titrated endogenous HRG-7 and HRG-7PR(D90A/D318A) expressed in the hrg-7(tm6801) background. Membranes were probed with polyclonal anti-HRG-7 antibody and then incubated with HRP-conjugated anti-rabbit secondary antibody. Irrelevant lanes were removed from the image. Unprocessed blots are shown in Figure S6. Images are representative of two independent experiments.
