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WBPicture0000014564 Description: Figure S5. MYRF-2 exhibits cytoplasmic and nuclear localization.Related to Figure 5.A. Illustration of GFP knock-in allele (ybq46) of myrf-2(F21A10.2c), in which GFP is inserted after Ile190. GFP canlabel all myrf-2 isoforms.B. Whole mount immunostaining on GFP::myrf-2 knock-in (ybq46) animals using anti-GFP. DAPI, a DNA-bindingdye. Scale, 10 um.C. Illustration of GFP::myrf-2::HA minigene (ybqIs107), including the 7.5 kb sequence upstream of the ATG for myrf2 a- isoform. The myrf-2 cDNA is inserted after the ATG for F21A10.2a. GFP is inserted after Ile190, which is in a lessconserved region and close to the N-terminus. HA tag is inserted at the C-terminus of MYRF-2.D. Whole mount immunostaining using anti-HA on dual-transgene animals with myrf-2 minigene (ybqIs107) and panneuronally (rgef-1pro) expressed ER marker cytb-5.1::mCherry (ybqEx599), or Golgi marker aman-2::mCherry(ybqEx601). Red and green signals in specific ROIs are subject to co-localization test "Cocol 2" in ImageJ andPearson's coefficiency (R) are noted. Scale, 1 um.E. Fluorescent native GFP signals from GFP-tagged full-length MYRF-2 and N-MYRF-2(1-499aa), or theimmunostaining on HA tag inserted in C-MYRF-2(500-946aa), when expressed in HEK293 cells. MYRF-2 variants areco-transfected with ER::DsRed or Golgi::DsRed. Scale bars, 5 um.F. Protein extracts from N2 and myrf-2pro-GFP::myrf-2::HA minigene transgene (ybqIs107) are analyzed by WesternBlot. Two bands (140 kDa, 75 kDa) are detected by antibody against GFP.G. Illustration of myrf-2 transcription reporter. The promoter includes the 7.5 kb sequence upstream of the ATG formyrf-2 a- isoform. The NLS::tagRFP is inserted after the ATG for F21A10.2a.H. NLS::tagRFP signals from myrf-2pro-NLS::tagRFP transgene (ybqEx560). Upper image, whole body view of a L2animal; Lower left, head region; Lower middle, intestine; Lower right, seam cells. Scale, 10 um.I. Co-localization of unc-25pro-mCherry::Rab-3 (juIs236) and myrf-2pro-NLS::tagRFP (ybqEx560) in dual-transgeneanimals at late L1. Scale, 10 um.K. Whole mount immunostaining using anti-GFP on GFP::myrf-2 knock-in (ybq46) animals. The animals also carryunc-25pro-mCherry::Rab-3 (juIs236) to label DD/VDs. Scale, 10 um. For analysis of GFP::MYRF-2 expression, antiGFP signals are imaged, and nuclear signals in DDs are normalized by background signal intensity. Normalizedintensity for early L1 and late L1 animals are significantly different; t-test with Welch's correction (P=0.004);Mean+-SEM for late L1, 5.033+-1.020, n=10; Mean+-SEM for early L1, 1.153+-0.067, n=10.
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  Acknowledgment: Template: Reprinted from <Journal_URL>, <Article_URL>, Copyright <Publication_year>, with permission from <Publisher_URL>.
    Publication_year: 2017
    Article_URL: DOI id 10.1016/j.devcel.2017.03.022
    Journal_URL: DevelopmentalCell
    Publisher_URL: Elsevier
  Reference: WBPaper00051143