Journal: EMBO Reports

Article Title: UbcD1 regulates Hedgehog signaling by directly modulating Ci ubiquitination and processing

doi: 10.15252/embr.201643289

Figure Lengend Snippet: RNA isolation, reverse transcription, and real‐time qPCR

Article Snippet: For real‐time qPCR, 0.5 μg RNA was used for reverse transcription with ReverTra Ace qPCR RT Master Mix with gDNA Remover (TOYOBO, FSQ‐301).

Techniques: Isolation, Reverse Transcription

Journal: PLoS ONE

Article Title: Functional characterization of zebrafish orthologs of the human Beta 3-Glucosyltransferase B3GLCT gene mutated in Peters Plus Syndrome

doi: 10.1371/journal.pone.0184903

Figure Lengend Snippet: (A) The two zebrafish orthologs of B3GLCT show overall similar exonic arrangement. The number of each exon is located within each box and the size of the exon (in base pairs) is shown above each exon. The 5’ and 3’ UTRs are indicated preceding the first ATG and following the stop codon ( TAA/TAG ). White indicates the N-terminal signal sequence, light grey indicates the stem region and dark grey indicates the catalytic domain. The vertical black bar in exon 12 of each gene indicates the location of nucleotides encoding for the catalytic tri-aspartic acid residues. Horizontal lines underneath the zebrafish genes indicate previously annotated sequence and sequence identified in this study. (B) Schematic of genomic context for B3GLCT/b3glct. (C) Multiple species alignment of B3GLCT orthologs from human (NP_919299), mouse (NP_001074673), Xenopus (NP_001072551), and zebrafish. Blue bar indicates signal peptide, green indicates stem region and orange indicates catalytic core. Grey shading of amino acids indicates conservation. The DxD motif is boxed in red.

Article Snippet: The plasmids encoding for TALENs targeting exon 12 of b3glcta or b3glctb were digested using SmaI (New England Biolabs, Ipswich, MA, USA); the linearized plasmids were purified and used as a template for mRNA synthesis using the T7 RNA polymerase.

Techniques: Sequencing

Journal: PLoS ONE

Article Title: Functional characterization of zebrafish orthologs of the human Beta 3-Glucosyltransferase B3GLCT gene mutated in Peters Plus Syndrome

doi: 10.1371/journal.pone.0184903

Figure Lengend Snippet: (A) Schematic of b3glct genes indicating TALEN target sites (exon 1 and 12 for b3glcta and exon 12 for b3glctb , black arrows). The predicted protein product resulting from TALEN mediated disruption is shown. Editing events in the first exon of b3glcta are predicted to disrupt nearly the entire coding region of the transcript. For both b3glcta and b3glctb , editing in the 12 th exon is predicted to result in loss of most of the catalytic domain including the catalytic core and KDEL-like ER retention signal. Blue (SP)- Signal Peptide, Green (SR)- Stem Region, Orange (CD)- Catalytic Domain. (B) Images of zebrafish embryos at 5-dpf and adult zebrafish showing no gross morphological defects associated with loss of b3glct . (C) Functional evaluation of wild-type and mutant b3glct by in vitro β3-glucosyltransferase assays. Left panel- the endogenous β3-glucosyltransferase activity toward O -fucosylated TSR3 is dependent on the amount of protein in the wild type zebrafish homogenate; control reaction with 10 μg homogenate was performed using unmodified TSR3. Right panel- the endogenous β3-glucosyltransferase activity toward O -fucosylated TSR3 in the homogenate of double homozygous b3glct embryos is profoundly reduced compared with that in the wild type zebrafish homogenate; control reactions were performed using unmodified TSR3. Assays were performed in triplicate. Error bars indicate s.d.

Article Snippet: The plasmids encoding for TALENs targeting exon 12 of b3glcta or b3glctb were digested using SmaI (New England Biolabs, Ipswich, MA, USA); the linearized plasmids were purified and used as a template for mRNA synthesis using the T7 RNA polymerase.

Techniques: Functional Assay, Mutagenesis, In Vitro, Activity Assay

Journal: Nucleic Acids Research

Article Title: Ablation of Mto1 in zebrafish exhibited hypertrophic cardiomyopathy manifested by mitochondrion RNA maturation deficiency

doi: 10.1093/nar/gkab228

Figure Lengend Snippet: Western blotting analysis of mitochondrial proteins. ( A , C ) Twenty micrograms of total proteins from mutant and WT zebrafish were electrophoresed through a denaturing polyacrylamide gel, electroblotted and hybridized with antibodies for 5 subunits of OXPHOS (mitochondrion-encoding Nd1, Co2 and Atp8 and nucleus-encoding Sdha and Atp5a), Tufm, Tfam and Tom20 as a loading control. Quantification of levels of OXPHOS subunits ( B ) and other mitochondrial proteins ( D ). Average contents of Nd1, Co2, Atp8, Atp5a, Sdha, Tufm and Tfam were normalized to the average content of Tom20 in mutant and WT zebrafish. The values for the mutant zebrafish are expressed as percentages of the average values for the WT zebrafish. The calculations were based on three independent determinations. Graph details and symbols are explained in the legend to Figure .

Article Snippet: The antibodies used for this investigation were from Sigma [Mto1 (Sigma, HPA030232) and Gapdh (SAB2701826)], Abcam [Nd1 (ab74257), Sdha (ab151684), Atp5a (ab188107), Mtpap (ab154555) and Uqcrc2 (ab203832)] and Proteintech [Co2 (55070-1-AP), Atp8 (26723-1-AP), Ndufs1 (12444-1-AP), Cox5a (11448–1-AP), Atp5c (60284-1-Ig), Tfam (19998-1-AP), and Tufm (26730-1-AP) and Tom20 (1802-1-AP)].

Techniques: Western Blot, Mutagenesis

Journal: Nucleic Acids Research

Article Title: Ablation of Mto1 in zebrafish exhibited hypertrophic cardiomyopathy manifested by mitochondrion RNA maturation deficiency

doi: 10.1093/nar/gkab228

Figure Lengend Snippet: The synergic effects between Mto1 and Mtpap on the polyadenylation of mRNAs. ( A, B ) Immunoprecipitation analysis of MTO1 with MTPAP. HEK 293T cells transiently expressing with or without MTO1-FLAG were solubilized with a lysis buffer and lysate proteins were immuno-precipitated with immunocapture buffer (left) (input) and FLAG-antibody (right) (IP), respectively. Immunoprecipitates were analyzed by SDS-PAGE and Western blotting using anti-FLAG, anti-MTPAP and TOM20 antibodies, respectively. ( C ) Western blot analysis. Twenty micrograms of total proteins from mto1 −/− and WT zebrafish were electrophoresed through a denaturing polyacrylamide gel, electroblotted and hybridized with antibodies for Mtpap and Gapdh as a loading control. ( D ) Polyadenylation profiles of mitochondrial mRNAs upon mto1 −/− and WT zebrafish hearts. The 3′ termini of nd1, cytb , cox1 and cox3 mRNAs were assessed by RT-PCR amplifications from polyadenylated and oligoadenylated RNAs of mto1 −/− and WT zebrafish hearts and 3% agarose gel electrophoresis. Arrows indicated the positions of PCR products from the polyadenylated and oligoadenylated RNAs, respectively. ( E ) Quantification of poly(A) proportions of nd1, cytb , cox1 and cox3 transcripts in the WT and mto1 −/− zebrafish. ( F ) Poly(A) tail lengths from individually sequenced clones after 3’ end RACE analysis of nd1 transcripts in the mto1 −/− and WT zebrafish hearts. Graph details and symbols are explained in the legend to Figure .

Article Snippet: The antibodies used for this investigation were from Sigma [Mto1 (Sigma, HPA030232) and Gapdh (SAB2701826)], Abcam [Nd1 (ab74257), Sdha (ab151684), Atp5a (ab188107), Mtpap (ab154555) and Uqcrc2 (ab203832)] and Proteintech [Co2 (55070-1-AP), Atp8 (26723-1-AP), Ndufs1 (12444-1-AP), Cox5a (11448–1-AP), Atp5c (60284-1-Ig), Tfam (19998-1-AP), and Tufm (26730-1-AP) and Tom20 (1802-1-AP)].

Techniques: Immunoprecipitation, Expressing, Lysis, SDS Page, Western Blot, Reverse Transcription Polymerase Chain Reaction, Agarose Gel Electrophoresis, Clone Assay