Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Synapse-specific catecholaminergic modulation of neuronal glutamate release.

doi: 10.1073/pnas.2420496121

Figure Lengend Snippet: Fig. 3. Type I MNs OA receptors include OAMB, which is enriched in the Ib MN and needed to boost locomotion. (A) RNAseq shows expression of five OA receptors in type I MNs. The dashed line shows the maximum normalized counts of any gene observed in attp2 controls. Error bars are ± S.E.M (B) Example image of an NMJ showing a maximum projection confocal image of OAMB receptor staining (green) in a Ib and Is MN pair. Animals had the OAMB receptor CRISPR tagged with 10 copies of the V5 epitope. Staining was against the V5 epitope. (Scale bar, 5 μm.) (C) Bar graph shows the mean ± 95% CI of the mean OAMB staining levels (A.U. arbitrary units) per Ib and Is MNs. Each Ib and Is were paired on the same muscle (10 NMJs, 1 per animal). (**** P <0.0001 by the paired t-test). (D) Mean velocity (mm/s) of control larvae (attp2) (black, n = 15) and animals with OAMB knocked down in type I MNs (oambRNAi; blue, n = 14) crawling in the dark (black bar) followed by under 505 nm light (blue bar). Recording starts at 5 min., following an adjustment period. (E) Mean total displacement for each animal in (D). Attp2 (black, n = 15 animals), oambRNAi (blue, n =14 animals). Red points are mean ± S.E.M. (*** P <0.001; ** P <0.01; n.s., not significant by Student’s paired t-test).

Article Snippet: For OAMB imaging, rat anti- V5 epitope (Novus NBP2- 81037) was used at 1:500.

Techniques: Expressing, Staining, CRISPR, Control

Journal: Virology

Article Title: Rho GTPase activity modulates paramyxovirus fusion protein-mediated cell-cell fusion.

doi: 10.1016/j.virol.2006.01.033

Figure Lengend Snippet: Fig. 6. Flow cytometry analysis of F protein surface expression. BHK or Vero cells were mock-transfected or transfected with Hendra F or SV5 F and a GTPase or empty vector. Cell surface Hendra F or SV5 F was labeled with primary antibody specific to Hendra F or SV5 F and a FITC-conjugated secondary antibody. Cells were gently lifted from the plate with washing in 50 mM EDTA and fixed in 1% formaldehyde prior to analysis of fluorescence. Data shown are the average of three independent experiments, and error bars represent the standard deviation.

Article Snippet: Hendra F-transfected cells were incubated at 37 °C overnight because syncytia formation does not occur in the absence of G. Cells were prepared for flow cytometry as described previously (Horvath and Lamb, 1992) with the SV5 F primary monoclonal antibody F1a, provided by Richard Randall (University of St Andrews, Fife, UK) (Randall et al., 1987), or polyclonal rabbit antibodies generated against Hendra F, and the secondary antibody fluorescein isothiocyanate (FITC)-conjugated goat anti-mouse (for SV5 F) or antirabbit (for Hendra F) immunoglobulin G (Jackson ImmunoResearch Laboratories, West Grove, PA).

Techniques: Flow Cytometry, Expressing, Transfection, Plasmid Preparation, Labeling, Fluorescence, Standard Deviation

Journal: Virology

Article Title: Rho GTPase activity modulates paramyxovirus fusion protein-mediated cell-cell fusion.

doi: 10.1016/j.virol.2006.01.033

Figure Lengend Snippet: Fig. 7. Immunofluorescence of SV5 glycoprotein-induced syncytia in BHK cells. (A) Products of SV5 syncytia assays were fixed with 4% paraformaldehyde, permeabilized with 1% Triton X-100, and labeled with primary antibody to the Myc-tagged GTPase. The FITC-conjugated secondary antibody was applied together with rhodamine phalloidin to stain F-actin. Cells were mounted with a DAPI stain to the nuclei. Images were contrast enhanced. Arrows indicate structures described in the main text. (B) Examples of the nuclear arrangement and actin structure promoted by Cdc42V12 in Hendra or SV5 glycoprotein-induced syncytia.

Article Snippet: Hendra F-transfected cells were incubated at 37 °C overnight because syncytia formation does not occur in the absence of G. Cells were prepared for flow cytometry as described previously (Horvath and Lamb, 1992) with the SV5 F primary monoclonal antibody F1a, provided by Richard Randall (University of St Andrews, Fife, UK) (Randall et al., 1987), or polyclonal rabbit antibodies generated against Hendra F, and the secondary antibody fluorescein isothiocyanate (FITC)-conjugated goat anti-mouse (for SV5 F) or antirabbit (for Hendra F) immunoglobulin G (Jackson ImmunoResearch Laboratories, West Grove, PA).

Techniques: Immunofluorescence, Labeling, Staining

Journal: iScience

Article Title: Evolutionary analyses of the animal glycosyltransferase family 54 reveals two β1,4-N-acetylglucosaminyltransferase families

doi: 10.1016/j.isci.2025.113788

Figure Lengend Snippet: Enzymatic activity of mouse MGAT4A-F, chicken MGAT4C and MGAT4F, and medaka MGAT4C-rA, MGAT4C-rB, and MGAT4D (A) Proteins from MGAT4A/MGAT4B DKO HEK293 cells transfected with an empty vector (mock) or a plasmid for expression of mouse MGAT4A-F, chicken MGAT4C or MGAT4F, or medaka MGAT4C-rA, MGAT4C-rB, or MGAT4D were subjected to SDS-PAGE and blotted with anti-V5 antibody (upper) or anti-GAPDH antibody (lower). (B) Overlay of HPLC chromatograms for acceptor substrates and branched N -glycan products in MGAT4 reactions. Lysates of DKO cells transfected with each plasmid were reacted with fluorescence (PA, 2-aminopyridine)-labeled bi-antennary N -glycan (left) or tri-antennary N -glycan with β1,6-GlcNAc on α1,6-Man (MGAT5 product) (right). The reaction mixtures were analyzed by reversed phase HPLC to separate the substrates and the products. The elution positions of the substrates and the expected products are indicated by red arrows. (C) Specific activity of the lysates expressing each enzyme is shown ( n = 3, mean ± SD).

Article Snippet: The anti-V5-tag antibody (mouse, clone SV5-P-K; v5ab) was from Proteintech, and the anti-GAPDH (mouse, clone 6C5; MAB374) was from Merck Millipore.

Techniques: Activity Assay, Transfection, Plasmid Preparation, Expressing, SDS Page, Glycoproteomics, Fluorescence, Labeling

Journal: iScience

Article Title: Evolutionary analyses of the animal glycosyltransferase family 54 reveals two β1,4-N-acetylglucosaminyltransferase families

doi: 10.1016/j.isci.2025.113788

Figure Lengend Snippet: Enzymatic activity of chimeric mouse MGAT4D (A) Plasmid constructs used in this study. (B) Proteins from MGAT4A/MGAT4B DKO HEK293 cells transfected with an empty vector (mock) or a plasmid for expression of mouse (mm) MGAT4A, mmMGAT4D, mmMGAT4D-mmLec(A), mmMGAT4D-mmLec(B), medaka (ol) MGAT4D, or mmMGAT4D-olLec(D) were subjected to SDS-PAGE and blotted with anti-V5 antibody (upper) or anti-GAPDH antibody (lower). (C) Overlay of HPLC chromatograms for acceptor substrates and branched N -glycan products in MGAT4 reactions. Lysates of DKO cells transfected with each plasmid were reacted with fluorescence (PA, 2-aminopyridine)-labeled bi-antennary N -glycan (left) or tri-antennary N -glycan with β1,6-GlcNAc on α1,6-Man (MGAT5 product) (right). The reaction mixtures were analyzed by reversed phase HPLC to separate the substrates and the products. The elution position of the substrates and the expected products are indicated by red arrows. (D) Specific activity of the lysates expressing each enzyme is shown ( n = 3, mean ± SD).

Article Snippet: The anti-V5-tag antibody (mouse, clone SV5-P-K; v5ab) was from Proteintech, and the anti-GAPDH (mouse, clone 6C5; MAB374) was from Merck Millipore.

Techniques: Activity Assay, Plasmid Preparation, Construct, Transfection, Expressing, SDS Page, Glycoproteomics, Fluorescence, Labeling