Figure 3. Dual Localization of MYRF-1 in Cytoplasm and Nucleus(A) Illustration of
myrf-1pro-GFP::MYRF-1::FLAG minigene transgene.(B) Percentage of
myrf-1(
ju1121) mutants carrying
myrf-1 minigene (ybqIs13) (picked at L1) that developed into fertile adults. WT, wild-type.(C) Number of synapses is quantified for
myrf-1(
ju1121) carrying
myrf-1 minigene transgene (ybqIs13), labeled by
flp-13pro-GFP::Rab-3 (ybqIs47), and shown asmean +- SEM (t test: n.s., not significant). Number of animals analyzed is shown on each bar.(D) GFP signal from
myrf-1 minigene (ybqIs13) increased at late L1. Arrows, ventral cord neurons with GFP signal. GFP signal in soma normalized by backgroundsignal is shown as mean +- SEM (boxes) (t test: ***p < 0.001); whiskers, minimum/maximum values; n, number of images analyzed. (E) Co-localization of native GFP signal from GFP::MYRF-1 (ybqEx164) and
unc-25pro-mCherry::RAB-3 (juIs236). Square box, area with closer view of DD to theright; rectangular box, area with closer view of DD/VD to the right; dotted circles, DD or VD soma. Scale bar, 10 um.(F) Tissue-specific expression of
myrf-1 cDNA in
myrf-1(
ju1121) mutants. Genomic
myrf-1 (ybqEx55), 11.5 kb amplified from genomic DNA; DD
flp-13 (ybqEx94);DD/VD
unc-25 (ybqEx85); epidermis
dpy-7 (ybqEx16); body wall muscle
myo-3 (ybqEx93); pan-neuron
rgef-1 (ybqEx86). Arrows, dorsal DD synapses. Percentage of animals with rewired DDs is shown as mean +- SEM (t test: ***p < 0.001). The number of animals analyzed is shown on each bar. Scale bar, 10 mm.(G) Immunostaining of
myrf-1 minigene transgene (ybqIs13) using anti-GFP and anti-FLAG. DAPI was used as DNA-binding dye. Scale bar, 10 mm.(H) Immunostaining using anti-FLAG on dual-transgene animals with
myrf-1 minigene (ybqIs13) and pan-neuronally (
rgef-1pro) expressed ER marker cytb5.1::mCherry transgenes (ybqEx595); or Golgi marker
aman-2::mCherry (ybqEx597). Red and green signals in each region of interest are tests for co-localization;R, Pearson's coefficient. Scale bar, 1 um.(I) Protein extracts from N2,
myrf-1(+), and GFP-
myrf-1(+) transgene animals (ybqEx55, ybqIs13) were analyzed by western blot. Two bands (140 kDa and 75 kDa)were detected by anti-GFP.See also Figures S3 and S4.