Journal: Science Advances
Article Title: Decoding the molecular logic of rapidly evolving ZAD zinc finger proteins in Drosophila
doi: 10.1126/sciadv.ady7568
Figure Lengend Snippet: ( A ) Two paralogous ZAD-ZnF genes D19B and D19A are tandemly placed at the locus. ( B ) Western blot analysis of GFP-3xFLAG–tagged endogenous D19A and D19B proteins. Two- to 4-hour embryos from corresponding homozygous strains were used for the analysis. As a control, 2- to 4-hour yw embryos were used. ( C ) Measurement of the embryo hatching rate. Embryos from corresponding homozygous full-locus deletion mutant adults were used for the analysis. ( D ) Confocal images of His2Av-emiRFP670 in embryos from WT or homozygous D19B[ Δ 1] mutant adults. The maximum-intensity–projected images of His2Av-emiRFP670 are shown. ( E ) Measurement of the embryo hatching rate. Maternal supply of D19B is essential for the correct progression of embryogenesis. ( F ) Schematic of the full-length D19B protein. ( G ) AlphaFold3 prediction of the D19B ZAD dimer. Protein structure was visualized using UCSF ChimeraX . ( H ) Western blot analysis of GFP-3xFLAG–tagged endogenous D19B and WT/ΔZAD D19B rescue constructs reintroduced into the endogenous locus via recombinase-mediated cassette exchange (RMCE). Two- to 4-hour embryos from corresponding homozygous mutant adults were used for the analysis. As a control, 2- to 4-hour yw embryos were used. ( I ) Airyscan imaging of GFP-3xFLAG–tagged WT and ΔZAD D19B expressed from the endogenous locus. Images were taken ~15 min after entry into nc14. Embryos from corresponding homozygous strains were used for the analysis. The maximum-intensity–projected images are shown. ( J ) Measurement of embryo hatching rate. Embryos from corresponding homozygous RMCE strains were used for the analysis.
Article Snippet: Dynabeads Protein G magnetic beads (Invitrogen) were diluted by adding 10 μl of beads to 100 μl of blocking solution [PBS containing bovine serum albumin (BSA; 5 mg/ml)].
Techniques: Western Blot, Control, Mutagenesis, Construct, Imaging
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![( A ) Two paralogous ZAD-ZnF genes D19B and D19A are tandemly placed at the locus. ( B ) Western blot analysis of GFP-3xFLAG–tagged endogenous D19A and D19B proteins. Two- to 4-hour embryos from corresponding homozygous strains were used for the analysis. As a control, 2- to 4-hour yw embryos were used. ( C ) Measurement of the embryo hatching rate. Embryos from corresponding homozygous full-locus deletion mutant adults were used for the analysis. ( D ) Confocal images of His2Av-emiRFP670 in embryos from WT or homozygous D19B[ Δ 1] mutant adults. The maximum-intensity–projected images of His2Av-emiRFP670 are shown. ( E ) Measurement of the embryo hatching rate. Maternal supply of D19B is essential for the correct progression of embryogenesis. ( F ) Schematic of the full-length D19B protein. ( G ) AlphaFold3 prediction of the D19B ZAD dimer. Protein structure was visualized using UCSF ChimeraX . ( H ) Western blot analysis of GFP-3xFLAG–tagged endogenous D19B and WT/ΔZAD D19B rescue constructs reintroduced into the endogenous locus via recombinase-mediated cassette exchange (RMCE). Two- to 4-hour embryos from corresponding homozygous mutant adults were used for the analysis. As a control, 2- to 4-hour yw embryos were used. ( I ) Airyscan imaging of GFP-3xFLAG–tagged WT and ΔZAD D19B expressed from the endogenous locus. Images were taken ~15 min after entry into nc14. Embryos from corresponding homozygous strains were used for the analysis. The maximum-intensity–projected images are shown. ( J ) Measurement of embryo hatching rate. Embryos from corresponding homozygous RMCE strains were used for the analysis.](https://pub-med-central-images-cdn.bioz.com/pub_med_central_ids_ending_with_7871/pmc12947871/pmc12947871__sciadv.ady7568-f5.jpg)