Journal: Virology

Article Title: Identification of Multiple Potent Neutralizing and Non-Neutralizing Antibodies against Epstein-Barr Virus gp350 Protein with Potential for Clinical Application and as Reagents for Mapping Immunodominant Epitopes

doi: 10.1016/j.virol.2019.07.026

Figure Lengend Snippet: (A) SDS-PAGE analysis of anti-EBV gp350 antibodies purified from indicated hybridoma (HB) supernatants. (B) ELISA screening of HB supernatants for anti-gp350-specific antibodies. Soluble EBV gp350 protein was used as the target antigen at 0.5 μg/ml. m72A1 at 10 μg/ml and KSHV anti-gH/gl (54A1) were used as positive and negative (not shown) controls, respectively. Bound antibodies were detected using HRP-conjugated anti-mouse IgG (1:2,000). Twenty-three HB clones with ELISA signals two times greater than those of PBS control were considered to be positive/reactive to gp350. (C) Immunoblot analysis with gp350-transfected stable CHO lysate to determine specificity of anti-gp350-producing HB supernatants. (D) Flow cytometric analysis of surface expression of gp350 protein on gp350-expressing CHO cells. Cells were stained with indicated anti-gp350 mAbs (1:250), followed by secondary goat anti-mouse conjugated to AF488.

Article Snippet: Secondary antibodies: Horseradish peroxidase (HRP)-conjugated goat anti-mouse IgG for immunoblot or ELISA was purchased from Bio-Rad.

Techniques: SDS Page, Purification, Enzyme-linked Immunosorbent Assay, Clone Assay, Western Blot, Transfection, Expressing, Staining

Journal: Virology

Article Title: Identification of Multiple Potent Neutralizing and Non-Neutralizing Antibodies against Epstein-Barr Virus gp350 Protein with Potential for Clinical Application and as Reagents for Mapping Immunodominant Epitopes

doi: 10.1016/j.virol.2019.07.026

Figure Lengend Snippet: (A) Sequence comparison of murine (m72A1) and humanized (h72A1) 72A1. ClustalW alignment of heavy chain (i) and light chain (ii) variable region amino acid sequences. Regions of identical sequence are represented by *. Regions of similarity are represented by:. (B) ELISA comparison screening of m72A1 and h72A1 for anti-gp350-specificity. Soluble EBV gp350 protein was used as the target antigen at 0.5 μg/ml. m72A1 and h72A1 were serially diluted (5–0.062 μg/ml) and 1x phosphate buffered saline (PBS) was used as a negative control (data not shown). Bound h72A1 and m72A1 antibodies were detected using HRP-conjugated anti-mouse IgG and anti-human IgG (1:2,000) as relevant. (C) ELISA determining the reactivity of humanized 72A1 to murine IgG. Soluble EBV gp350 protein was used as the target antigen at 0.5 μg/ml. Plates were incubated with 10 μg/ml of m72A1 and h72A1, followed by three washes. Bound antibodies were detected using HRP-conjugated anti-mouse IgG or anti-human IgG (1:2,000). (D) Flow cytometric analysis of m72A1 and h72A1 gp350 specificity. CHO wild-type cells and gp350-expressing CHO cells were stained with m72A1 and h72A1, followed by secondary goat anti-mouse or anti-human conjugated to AF488. Unstained cells and cells stained with secondary goat anti-mouse or anti-human conjugated to AF488 alone were used as negative controls. 2° represents secondary antibody.

Article Snippet: Secondary antibodies: Horseradish peroxidase (HRP)-conjugated goat anti-mouse IgG for immunoblot or ELISA was purchased from Bio-Rad.

Techniques: Sequencing, Enzyme-linked Immunosorbent Assay, Negative Control, Incubation, Expressing, Staining

Journal:

Article Title: Phospholipase D2 Is Localized to the Rims of the Golgi Apparatus in Mammalian Cells

doi: 10.1091/mbc.02-04-0059

Figure Lengend Snippet: PLD2 localization with different organelles in rat NRK cells. Cells were prepared for immunofluorescence microscopy and incubated with rabbit PLD2 antibody PLD2-27 (A, D, G, and J) and costained with monoclonal antibodies to the ER marker BiP (B); the transferrin receptor (E), a marker of early endosomes and the plasma membrane; and lgp120 (H), a late endosome/lysosomal marker. (K) Cells were stained with caveolin-1, a marker for caveoli; the arrow corresponds to caveolin-1 localized to the plasma membrane. Images were merged to determine overlap between PLD2 (red) and the respective marker proteins (green; C, F, I, and L). Areas of maximal overlap are yellow. (L) Inset, enlargement of the region of overlap between PLD2 and caveolin-1 in the perinuclear Golgi apparatus. The arrow indicates areas where PLD2 puncta (red) either overlap completely with caveolin-1 (green) or are directly adjacent. Images are from projected Z-series. Bars, 10 μm.

Article Snippet: Purified monoclonal anti-rat transferrin receptor antibodies were purchased from Cedarlane Laboratories (Hornby, Ontario, Canada).

Techniques: Immunofluorescence, Microscopy, Incubation, Marker, Staining

Journal: PLoS ONE

Article Title: Monocytes-Derived Macrophages Mediated Stable Expression of Human Brain-Derived Neurotrophic Factor, a Novel Therapeutic Strategy for NeuroAIDS

doi: 10.1371/journal.pone.0082030

Figure Lengend Snippet: 5×10 5 HTB-10, HTB-11 and CHME-5 cells were subcultured in 25 cm 2 tissue culture flask 24 hrs before transduction, then removed the media, DPBS washed cells twice, added 0.5 mL vector suspension (1×10 7 IU/mL, MOI = 10, for HTB-11, MOI = 1) containing 8 µg/mL polybrene, and incubated at 37°C for 2 hours with gentle mixing every 15 minutes. Then aspirated vector suspension and added 4 mL of fresh growth medium and incubated at 37°C with 5% CO 2 . The medium was replaced 24 hours post-infection and observed cells on day 3 post-infection under fluorescence microscope (Nikon Eclipse TE2000-U). (A) Photomicrographs of LV-transduced neuronal cell lines showing GFP exression. NL: Normal Light. FL: Fluorescent Light. (B) Immunofluorescent staining of GFP in neuronal cell lines. NL: Normal Light. GFP: overlay of green fluorescence. TRITC: overlay of red fluorescence. (C) Moderate to high levels of secreted hBDNF in LV-transduced neuronal cells conditioned media quantified by ELISA. Capture and detection antibodies were rabbit anti-human BDNF, goat anti-human IgG-Biotin respectively. (D) Stable expression of hBDNF detected from transduced neuronal cells. ELISA was performed every 5 passages for 20 passages to assess long-term stable expression of hBDNF gene. (E) Accumulative expression of hBDNF detected in LV-transduced HTB-11 cells.

Article Snippet: The secondary antibody used was Donkey anti-Goat IgG with TRITC (Rockland, USA).Cells were visually examined and documented using an inverted fluorescence microscope (Nikon Eclipse TE2000-U) with a digital camera attachment.

Techniques: Transduction, Plasmid Preparation, Incubation, Infection, Fluorescence, Microscopy, Staining, Enzyme-linked Immunosorbent Assay, Expressing

Journal: iScience

Article Title: Innate mechanism of mucosal barrier erosion in the pathogenesis of acquired colitis

doi: 10.1016/j.isci.2023.107883

Figure Lengend Snippet:

Article Snippet: Muc2 or lysozyme was visualized with 0.4 μg/ml of Texas Red-conjugated goat anti-rabbit IgG secondary antibodies (Santa Cruz Biotechnology); Neu3 was visualized with 0.4 μg/ml of FITC-conjugated goat anti-rabbit IgG secondary antibodies (Santa Cruz Biotechnology); αTubulin was visualized with 0.4 μg/ml of Texas Red-conjugated rabbit anti-goat IgG secondary antibodies (Santa Cruz Biotechnology); and cathepsin G or biotinylated lectins were visualized with 1 μg/ml of FITC-conjugated streptavidin (Vector Laboratories).

Techniques: Plasmid Preparation, Virus, Recombinant, Western Blot, Protease Inhibitor, Staining, Labeling, Reverse Transcription, SYBR Green Assay, Bacteria, Software, Imaging

Journal: Journal of Biological Chemistry

Article Title: Topogenesis of Peroxisomal Membrane Protein Requires a Short, Positively Charged Intervening-loop Sequence and Flanking Hydrophobic Segments

doi: 10.1074/jbc.m003304200

Figure Lengend Snippet: FIG. 2. Functional and topogenic regions of PMP34. C-terminally HA- tagged or GFP-fused PMP34 and its vari- ants were verified for intracellular local- ization in CHO-K1. A, constructs of deletion mutants of PMP34. DN30HA, PMP34-HA with deletion of N-terminal residues from 1 to 30; 204HA, HA-tagged PMP34 with residues 1–204; 204GFP, PMP34 comprising residues 1–204 fused with GFP. Others likewise representing respective constructs were indicated. Numbers in box represent the positions of transmembrane segments; L1–L5 desig- nate the intervening-loop region between two flanking TMs. Peroxisomal targeting activity of each variant verified (see be- low) was shown: 1, active; 1/2, partially active; 2, inactive. B, PMP34 variants represented in A were expressed in CHO- K1. a and b, DN30HA; c and d, DN125HA; e and f, DN186HA; g and h, DN125GFP; i and j, DN186GFP; k, DN204HA; l, 186HA; m and n, 204HA; o and p, 204GFP. C- terminally HA-tagged PMP34 variants were verified for peroxisomal localization by immunostaining using mouse (a, c, e, and m) and rabbit (k and l) anti-HA anti- body and FITC-labeled second antibody, where peroxisomes were assessed by anti- Pex14p antibody and Texas Red-labeled second antibody (b, d, f, h, j, n, and p). PMP34 truncation mutants fused with GFP were verified by GFP fluorescence (g, i, and o). Arrowheads indicate PMP34- positive particles, positive in expressed PMP34-variants, that were absent from Pex14p. Original magnification, 3630; bar, 20 mm. C, transmembrane topology of GFP fusion proteins, DN125GFP and 204GFP, was determined. CHO-K1 cells expressing DN125GFP (a and b) and 204GFP (c and d) were fixed, then treated with 25 mg/ml digitonin. Localization and membrane orientation were verified by GFP fluorescence (a and c) and immuno- fluorescence staining of GFP with anti- GFP antibody and Texas Red-labeled sec- ond antibody (b and d). Bar, 20 mm.

Article Snippet: Antigen-antibody complexes were detected under a Carl Zeiss Axioskop FL microscope, using fluorescein isothiocyanate (FITC)-labeled sheep anti-mouse antibody (Amersham Pharmacia Biotech, Tokyo, Japan), FITC-labeled sheep anti-rabbit immunoglobulin (Ig) G antibody (Cappel), or Texas Red-labeled goat antibodies to guinea pig IgG (Vector Laboratories) and rabbit IgG (Leinco Technologies).

Techniques: Functional Assay, Construct, Activity Assay, Variant Assay, Immunostaining, Labeling, Fluorescence, Expressing, Membrane, Staining

Journal: Journal of Biological Chemistry

Article Title: Topogenesis of Peroxisomal Membrane Protein Requires a Short, Positively Charged Intervening-loop Sequence and Flanking Hydrophobic Segments

doi: 10.1074/jbc.m003304200

Figure Lengend Snippet: FIG. 5. Coordinated function of the membrane targeting se- quence and transmembrane segments. A, constructs of the loop region and transmembrane segments (loop plus TM) fused with GFP. B, intracellular localization of the (loop plus TM)-GFP fusion protein. a and b, 86/204GFP; c and d, 30/204GFP; e, 125/273GFP; f, 86/273GFP. Each construct was expressed in CHO-K1 cells and detected by GFP fluorescence (a, c, e, and f). Cells expressing 86/204GFP were also stained using anti-malate dehydrogenase antibody and Texas Red- labeled second antibody (b); peroxisomes in 30/204GFP-expressing cells were assessed by anti-Pex14p antibody (d). Bar, 20 mm.

Article Snippet: Antigen-antibody complexes were detected under a Carl Zeiss Axioskop FL microscope, using fluorescein isothiocyanate (FITC)-labeled sheep anti-mouse antibody (Amersham Pharmacia Biotech, Tokyo, Japan), FITC-labeled sheep anti-rabbit immunoglobulin (Ig) G antibody (Cappel), or Texas Red-labeled goat antibodies to guinea pig IgG (Vector Laboratories) and rabbit IgG (Leinco Technologies).

Techniques: Membrane, Construct, Fluorescence, Expressing, Staining, Labeling