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: 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

Journal: bioRxiv

Article Title: Gαi2-Mediated Regulation of Microtubules Dynamics and Rac1 Activity Orchestrates Cranial Neural Crest Cell Migration in Xenopus

doi: 10.1101/2023.09.07.556733

Figure Lengend Snippet: A-B. Immunofluorescence against acetylated tubulin (green) and actin (red) in control and Gαi2 morphant explants to localize stable microtubules. Under Gαi2 knockdown conditions acetylated tubulin increases towards the leading edge. C-D. Western blot performed to measure the quantity of acetylated tubulin under control and Gαi2 knockdown conditions, showing an increase in the acetylated tubulin amount in morphant conditions. E. Immunofluorescence against endogenous Gαi2 and α-tubulin proteins to study subcellular localization. F. Protein colocalization was analyzed using Image’s JACoP software whose Pearson coefficient was 0.743, while the overlap coefficient was 0.779. G. Coimmunoprecipitation assays show that Gαi2 is interacting with α-tubulin. normal IgG: non-related IgG; IPP V5: immunoprecipitation conjugated to V5 antibody; input: total lysate. n total = 3 lysates per condition. H-I. Coimmunoprecipitation assays show that Gαi2 interacts with EB1 and EB3, plusTip proteins that regulate microtubules dynamic. W/A: Without antibody; normal IgG: non-related IgG; IPP V5: immunoprecipitation conjugated to V5 antibody; input: total lysate; elu: eluted. n total = 3 lysates per condition. J-M. Cranial NC explants were extracted from stage 17 Xenopus embryos, previously injected with Gαi2MO. The cell dispersion in each condition was evaluated during 10 hours through timelapse. All embryos were co-injected with H2B-Cherry and membrane GFP for individual cell monitoring and quantification of migration. Four conditions were evaluated: Control, Gαi2MO, Gαi2MO + 65 nM Nocodazole and Control + 65 nM Nocodazole. Under each representative image, Delaunay triangulation is shown in each condition at the final time (10 hours). N. The rate between the average areas corresponding to the final time were plotted at 10 hours. Nocodazole can rescue Gαi2 morphant phenotype and does not affect control migration. Significance was assessed with a Mann-Whitney test for non-parametric data (****, p<0.0001). Error bars: s.e.m. 10X magnification. Control n = 21 explants; Gαi2MO n = 24 explants; Gαi2MO + 65 nM Nocodazole n = 36 explants; Control + 65 nM Nocodazole n = 30 explants; 3 experiments.

Article Snippet: Western blots were performed as described previously (Kuriyama and Mayor, 2009) using the following antibodies: anti Gαi2 (Santa Cruz, L-5; 1:1000); anti GAPDH (Novus Biologicals, 1D4; 1:1000); anti V5-probe (Santa Cruz, sv5-pk; 1:5000); anti acetylated tubulin (Sigma, 611B1; 1:1000); anti α-tubulin (Sigma, T9026; 1:500); anti GFP (ThermoFisher, C163; 1:500); anti EB1 (Sigma, E3406; 1:100); mouse IgG HRP (Jackson ImmunoResearch Laboratories, 1:10000); rabbit IgG HRP (Jackson ImmunoResearch Laboratories, 1:10000).

Techniques: Immunofluorescence, Control, Knockdown, Western Blot, Software, Immunoprecipitation, Injection, Dispersion, Membrane, Migration, MANN-WHITNEY