Fig. S7

Figure S7. Expression pattern and phylogenetic analysis of Nipsnap1 and Nipsnap2in Zebrafish,Related to Figure 7A-B.ClustalW alignment of Nipsnap1 (A) and Nipsnap2/Gbas (B) amino acid sequences from zebrafish (Danio rerio, accession NP_571108 and NP_571109, respectively) with the respective human and mouse sequences. Identical residues are boxed in black. The N-terminal 23 amino acids of zebrafish Nipsnap1 are predicted to act as a MLS (MitoProt II) (red line). The DABB1 domain (green boxed region)and the DABB2/Nipsnap domain (blue boxed region) are highly conserved with amino acid identities of ≥ 90%, indicating a similar function across species. C. Temporal expression pattern of nipsnap1 andnipsnap2. The graph shows the fold change in transcript relative to beta-actin in whole zebrafish embryos from 1 cell to 7 dpf. Error bars indicate mean ± SEM D. Spatial expression pattern of nipsnap2 across the different development stages of zebrafish as demonstrated by whole-mount in situ hybridization at the indicated stages. All embryos were in lateral view. Scale bar -200 μm. E. Immunblotting for Nipsnap1 and Nipsnap2 in lysates from different tissues of adult zebrafish. F. Manipulations of the zf nipsnap1gene. Schematic description of the exon (black boxes) and intron (black lines) structure of the nipsnap1 gene. Scissors indicate the sgRNA1 used to target the 1st exon to make the nipsnap1 KO embryos (nipsnap-/-). A chromatogram showing the genotype of then nipsnap1 sa14357 mutant allele (nipsnap1mutant) is shown. The change in allele from T to A has been highlighted. G. Representative immunoblotting images of Nipsnap1 and β-tubulin on whole embryo lysates of nipsnap1 mutant, rescue and WT embryos at 3dpf. β-tubulin serves as the loading control. H. Representative FACS plot showing oxidative stress in Controls (WT), nipsnap1-/- embryos and nipsnap1 mutant embryos at 3 dpf using the CellROX reagent. H2O2 was added to the water for 1 h as a positive control. I. Quantification of the Th1 signal intensities from blots in (Fig 7G) normalized to WT signal intensity. Error bars indicate mean ± SEM. *p < 0.05, **p < 0.005, ***p < 0.0005, **** p < 0.00005, unpaired Students t-test. J.Qualitative analysis of the plate used for zebrabox experiments with L-DOPA added. Top 6 wells contain WT larvae, bottom six wells contain nipsnap1 mutant larvae. Plate was used for swimming assay earlier and then kept overnight to see oxidation of L-Dopa by visualizing the change in color.