Journal: PLOS One

Article Title: Activated human B cells produce phospholipase D4-containing extracellular vesicles

doi: 10.1371/journal.pone.0329832

Figure Lengend Snippet: (A) Immunofluorescence microscopy displaying localization of PLD4 (red) at TGN46-tagged trans Golgi networks (green) in resting B cells and translocation of PLD4 from trans Golgi networks in CpG/anti-IgG + M stimulated B cells, compared to the control cells. (B) Immunofluorescence microscopy shows enhanced expression of PLD4 (red) and co-localization of PLD4 and CD63 (green) in CpG/anti-IgG + M stimulated B cells, compared to the control cells. Cell membranes stained with MemBrite appeared thicker in CpG/anti-IgG + M stimulated B cells than in the control cells. (C) Statistical analysis revealing enhanced PLD4 expression in each CpG/anti-IgG + M stimulated B cell compared to the control cells. Bar; 10 μm, **P < 0.01, ***P < 0.001.

Article Snippet: The following antibodies were used: Recombinant human anti-human PLD4 therapeutic antibody biotinylated (TAB-759CL-Biotin; Creative Biolabs, NY, USA); rabbit anti-PLD4 polyclonal antibody (HPA051512; Atlas Antibodies, Bromma, Sweden); rat anti-CD9 monoclonal antibody (017−28211; FUJIFILM Wako, Osaka, Japan); FITC mouse anti-human CD63 antibody (353005; BioLegend, San Diego, CA, USA); PerCP/Cyanine5.5 mouse anti-human CD19 antibody (302230; BioLegend); Mouse anti-human CD19 antibody (302202; BioLegend); rabbit anti-TGN46 polyclonal antibody (13573–1-AP; Protein Tech, IL, USA).

Techniques: Immunofluorescence, Microscopy, Translocation Assay, Control, Expressing, Staining

Journal: Life Science Alliance

Article Title: A biallelic variant in GORASP1 causes a novel Golgipathy with glycosylation and mitotic defects

doi: 10.26508/lsa.202403065

Figure Lengend Snippet: (A) Surface staining of fluorophore-conjugated lectin WGA on non-permeabilized primary fibroblasts showing the reduced glycosylation of surface membranes in patient’s cells, which is rescued upon the re-expression of GRASP65-GFP. Scale bar: 50 μm. Quantification is presented as a percentage of the mean integrated signal density of control values ± SEM. * P ≤ 0.05, one-way ANOVA. (B) Surface staining of fluorophore-conjugated lectin MAAII on non-permeabilized primary fibroblasts showing the reduced glycosylation of surface membranes in patient’s cells, which is rescued upon the re-expression of GRASP65-GFP. Scale bar: 50 μm. Quantification is presented as a percentage of the mean integrated signal density of control values ± SEM. * P ≤ 0.05, one-way ANOVA. (C) Western blot analysis of LAMP-1, LAMP-2, and TGN46 normalized on GAPDH in control and patient’s fibroblasts showing a shift of the bands in patient’s fibroblasts. (D) Two-dimensional electrophoresis of the mucin core1 O-glycosylated apolipoprotein C-III (apoC-III) showing a reduced ratio of sialylation in the patient’s sample. Yellow square = N-acetylgalactosamine; yellow circle = galactose; purple diamond = sialic acid.

Article Snippet: Primary antibodies used were as follows: GRASP65 (NP2-02665, 1:1,000; Novus, and MA5-34658, 1:1,000; Invitrogen), GRASP55 (GORASP2, 10598-1-AP, 1:2,000; Proteintech), GM130 (610823, 1:1,000; BD Biosciences), LAMP-1 (9091S, 1:1,000; Cell Signaling), LAMP-2 (ab25631, 1:500; Abcam), TGN46 (APH500G, 1:1,000; Bio-Rad), GAPDH (60004-1-1g, 1:10,000; Proteintech).

Techniques: Staining, Glycoproteomics, Expressing, Control, Western Blot, Electrophoresis