Journal: The Journal of Biological Chemistry

Article Title: Basigin Interacts with Plasmodium vivax Tryptophan-rich Antigen PvTRAg38 as a Second Erythrocyte Receptor to Promote Parasite Growth *

doi: 10.1074/jbc.M116.744367

Figure Lengend Snippet: Identification of putative erythrocyte surface proteins interacting with PvTRAg38 by MudPIT analysis Proteins were identified after LC-MS/MS analysis following in solution digestion of GST-PvTRAg38 pulled down proteins.

Article Snippet: Materials The following were commercially obtained: human basigin cDNA clone, mammalian expressed recombinant histidine-tagged CD59 (Sino Biological, Beijing, China); human erythrocyte 55-kDa membrane protein (GeneCopoeia, Rockville, MD); Alexa Fluor 488-conjugated goat anti-mouse and anti-rabbit IgG, proofreading Pfx polymerase enzyme (Invitrogen Life Technologies, Inc.); sulfo-SBED-biotin Label transfer cross-linker (sulfosuccinimidyl-2-[6-(biotin amide)-2-( p -azidobenzamido)hexanoamido] ethyl-1,3′-dithiopropionate) (Thermo Scientific, Rockford, IL); GeneJET gel extraction kit (Thermo Scientific, Waltham, MA); HEK-293 cells (American Type Culture Collection (ATCC), Manassas, VA); RPMI 1640 medium, hypoxanthine, penicillin/streptomycin, fetal calf serum, glutamine, glutaraldehyde, poly- l -lysine, monoclonal antibodies against basigin (Clone MEM-M6); His 6 and GST (Sigma); Lipofectamine® 2000, synthetic peptides (Thermo Fisher Scientific, GmbH, Germany); mouse monoclonal antibodies DL6 (Santa Cruz Biotechnology, Dallas, TX); HBS-EP buffer (degassed and ready to use 0.01 m HEPES, pH 7.4, 0.15 m NaCl, 3 m m EDTA, 0.005% v/v surfactant P20); protein marker standards for GPC (GE Healthcare); Matchmaker Gold Systems (Clontech); and trypsin (Promega Corp., Madison, WI).

Techniques: Mass Spectrometry

Journal: The Journal of Biological Chemistry

Article Title: Basigin Interacts with Plasmodium vivax Tryptophan-rich Antigen PvTRAg38 as a Second Erythrocyte Receptor to Promote Parasite Growth *

doi: 10.1074/jbc.M116.744367

Figure Lengend Snippet: Binding of PvTRAg38 to basigin. A, solid phase ELISA. Increasing concentrations (0–2.5 μm) of histidine-tagged basigin, 55-kDa EMP, or CD59 were added to the wells of an ELISA plate already coated with 50 nm GST-tagged PvTRAg38 or untagged thioredoxin (inset). The plate was developed with anti-His6 monoclonal antibody as described in text. Mean ± S.D. value of absorbance from three experiments is plotted. B, SPR analysis of basigin interaction with PvTRAg38. The recombinant basigin was immobilized on the cell of CM5 chip by amine coupling method. Five different concentrations of recombinant PvTRAg38 (100–500 nm) were injected at a flow rate of 30 μl/min over the surface of immobilized basigin. Curve fit (Langmuir 1:1 model) sensograms show dose-dependent response of PvTRAg38 binding with basigin. C, direct interaction between basigin and PvTRAg38 by Label transfer assay. Recombinant histidine-tagged basigin was incubated with recombinant GST-tagged PvTRAg38 (labeled with trifunctional Sulfo-SBED cross-linker) and processed as in Fig. 1A. The eluate was washed and resolved on 15% SDS-PAGE and transferred to nitrocellulose membrane. The blot was probed with streptavidin-HRP. Lane 1, GST-tagged PvTRAg38 and histidine-tagged basigin after Label transfer. Lane 2, labeled GST-tagged PvTRAg38. Sizes of molecular weight marker proteins are shown on right-hand side. D, specificity of binding of PvTRAg38 to basigin by competition assay. Increasing concentrations of histidine-tagged PvTRAg38 (0–2 μm) were incubated for 2 h with recombinant-untagged basigin immobilized on an ELISA plate well. After washing, a fixed concentration of GST-tagged PvTRAg38 was added to the wells, and the bound protein was detected with GST monoclonal antibody as described in the text. Binding in the absence of histidine-PvTRAg38 was taken as percentage control for the rest of the concentrations. The mean value of three independent experiments is plotted with S.D. E, specificity of binding between basigin and PvTRAg38 by antibody inhibition assay using anti-PvTRAg38 antibodies. Different dilutions of anti-PvTRAg38 antibody were incubated with recombinant GST-tagged PvTRAg38 immobilized on an ELISA plate well for 2 h. After washing, a fixed concentration of histidine-tagged basigin was added to the wells. The bound histidine-tagged basigin was detected using monoclonal anti-His6 antibodies as described in the text. The mean value of three independent experiments is plotted with S.D. F, specificity of binding between basigin and PvTRAg38 by antibody inhibition assay using anti-basigin antibodies. Different dilutions of polyclonal anti-basigin antibody were incubated with recombinant histidine-tagged basigin immobilized on an ELISA plate well for 2 h. After washing, a fixed concentration of GST-tagged PvTRAg38 was added to the wells, and bound recombinant GST-tagged PvTRAg38 was detected by using anti-GST antibodies as described in the text. The mean value of three independent experiments is plotted with S.D.

Article Snippet: Materials The following were commercially obtained: human basigin cDNA clone, mammalian expressed recombinant histidine-tagged CD59 (Sino Biological, Beijing, China); human erythrocyte 55-kDa membrane protein (GeneCopoeia, Rockville, MD); Alexa Fluor 488-conjugated goat anti-mouse and anti-rabbit IgG, proofreading Pfx polymerase enzyme (Invitrogen Life Technologies, Inc.); sulfo-SBED-biotin Label transfer cross-linker (sulfosuccinimidyl-2-[6-(biotin amide)-2-( p -azidobenzamido)hexanoamido] ethyl-1,3′-dithiopropionate) (Thermo Scientific, Rockford, IL); GeneJET gel extraction kit (Thermo Scientific, Waltham, MA); HEK-293 cells (American Type Culture Collection (ATCC), Manassas, VA); RPMI 1640 medium, hypoxanthine, penicillin/streptomycin, fetal calf serum, glutamine, glutaraldehyde, poly- l -lysine, monoclonal antibodies against basigin (Clone MEM-M6); His 6 and GST (Sigma); Lipofectamine® 2000, synthetic peptides (Thermo Fisher Scientific, GmbH, Germany); mouse monoclonal antibodies DL6 (Santa Cruz Biotechnology, Dallas, TX); HBS-EP buffer (degassed and ready to use 0.01 m HEPES, pH 7.4, 0.15 m NaCl, 3 m m EDTA, 0.005% v/v surfactant P20); protein marker standards for GPC (GE Healthcare); Matchmaker Gold Systems (Clontech); and trypsin (Promega Corp., Madison, WI).

Techniques: Binding Assay, Enzyme-linked Immunosorbent Assay, Recombinant, Injection, Incubation, Labeling, SDS Page, Molecular Weight, Marker, Competitive Binding Assay, Concentration Assay, Inhibition

Journal: iScience

Article Title: Chimpanzee adenovirus-mediated multiple gene therapy for age-related macular degeneration

doi: 10.1016/j.isci.2023.107939

Figure Lengend Snippet: In vivo expression of eGFP, PEDF, sFlt-1, and sCD59 following IVT administration of AdCs (A) Representative fundus images of the retina from individual mice following IVT injection of 1.5×10 9 vp and 7.5×10 9 vp AdC68-eGFP. The eGFP signal could be detected from 48 h to 35 days post-injection. The dotted circles represent the edge of mouse retina. (B–D) Assessment of PEDF, sFlt-1, and sCD59 mRNA expression in retina-choroid-sclera complexes isolated from 10 mice. In each mouse, one eye was injected with AdC68-PFC (five mice for 1.5×10 9 vp and five mice for 7.5×10 9 vp) whereas the contralateral, un-injected eye served as control (only five eyes were used for analysis). At 4 days post-injection, RNA was purified from the retina-choroid complexes and real-time qPCR was conducted. Absolute number of mRNA copies were calculated using the standard curve method. (E–H) Images of western blot and quantification of the PEDF, sFlt-1, and sCD59 protein amount expressed in retina-choroid complexes of five mice. In each mouse, one eye was injected with AdC68-PFC (7.5×10 9 vp) whereas the contralateral, un-injected eye served as control. Total protein was obtained from retina-choroid-sclera complexes isolated from AdC68-PFC-treated (7.5×10 9 vp) and un-injected eyes 7 days post-injection. Antibodies against GAPDH were used for the internal control. The relative expression of PEDF, sFlt-1, and sCD59 in the un-injected eyes was set to 1. Data are expressed as mean ± SEM, and analyzed using one-way ANOVA multiple comparisons with Tukey’s method among groups in (B) and Student’s t test (two-tailed) in (C) (∗p < 0.05, ∗∗p < 0.01). PEDF, pigment epithelium-derived factor; sFlt-1, soluble fms-like tyrosine kinase-1; sCD59, soluble forms of CD59; IVT, intravitreal.

Article Snippet: PVDF membranes were blocked with 5% milk in PBST (PBS+Tween-20) for 2 h at RT and then target proteins were detected by specific primary antibodies including PEDF (1:500, 11104-RP02, Sino Biological, China), VEGFR1 (1:500, AF7748, Affinity, US), sCD59 (1:500, 12474-RP02, Sino Biological, China), Erk1/2 p44/42 (1:1000, 9102, Cell Signaling Technology, US), Erk1/2 phospho-p44/42 (T202/Y204) (1:1000, 9106, Cell Signaling Technology, US), p38 MAPK(1:1000, 8690, Cell Signaling Technology, US), Phospho-p38 MAPK (Thr180/Tyr182) (1:1000, 4631, Cell Signaling Technology, US), ICAM-1 (1:1000, 10831-1-AP, Proteintech, US), VCAM-1 (1:200, sc-13160 , Santa Cruz biotechnology, US).

Techniques: In Vivo, Expressing, Injection, Isolation, Control, Purification, Western Blot, Two Tailed Test, Derivative Assay

Journal: iScience

Article Title: Chimpanzee adenovirus-mediated multiple gene therapy for age-related macular degeneration

doi: 10.1016/j.isci.2023.107939

Figure Lengend Snippet:

Article Snippet: PVDF membranes were blocked with 5% milk in PBST (PBS+Tween-20) for 2 h at RT and then target proteins were detected by specific primary antibodies including PEDF (1:500, 11104-RP02, Sino Biological, China), VEGFR1 (1:500, AF7748, Affinity, US), sCD59 (1:500, 12474-RP02, Sino Biological, China), Erk1/2 p44/42 (1:1000, 9102, Cell Signaling Technology, US), Erk1/2 phospho-p44/42 (T202/Y204) (1:1000, 9106, Cell Signaling Technology, US), p38 MAPK(1:1000, 8690, Cell Signaling Technology, US), Phospho-p38 MAPK (Thr180/Tyr182) (1:1000, 4631, Cell Signaling Technology, US), ICAM-1 (1:1000, 10831-1-AP, Proteintech, US), VCAM-1 (1:200, sc-13160 , Santa Cruz biotechnology, US).

Techniques: Virus, Recombinant, Transfection, Protease Inhibitor, Plasmid Preparation, Gel Extraction, Software

Journal: Nature Communications

Article Title: Contemporary HIV-1 consensus Env with AI-assisted redesigned hypervariable loops promote antibody binding

doi: 10.1038/s41467-024-48139-x

Figure Lengend Snippet: For each panel, comparisons of IC50 values for short and long HV loops are shown for representative antibodies: PGT145 ( A ) and VRC26.25 ( B ), two V2 apex-targeting antibodies whose epitopes are close to V2HV loops; 10-1074 ( C ) and the PGT135 ( D ), two glycan supersite antibodies whose epitopes are close to V1HV loops; VRC01 ( E ) and 3BNC117 ( F ), two CD4 binding site antibodies with epitopes close to the V5HV loops. The HV loop nearest to the epitope of each antibody is shown in bold font. The box plots depicted the median and 25th/75th percentiles of the distribution, with the minima and maxima as whiskers. The p -values from non-paired, non-parametric one-tailed Mann-Whitney U tests are provided. IC50 values equal to or greater than 100 μg/ml are grouped (upper limit of antibody neutralization measurements in the CATNAP database). Source data for all panels are provided in the Source Data files.

Article Snippet: 293 T cells were transfected with 1 ug of plasmid DNA encoding an HIV-1 env gene (PolyJet™ In Vitro DNA Transfection Reagent (SignaGen Laboratories, Cat. No. SL100688) in 6-well plates and harvested at 48 h. Cells were split into separate staining reactions for monoclonal antibodies 3BNC117 (Cat. No. ARP-12474), 10-1074 (Cat. No. ARP-12477), 10E8 (Cat. No. ARP-12294), 447-52D (Cat. No. ARP-4030, all from BEI resources), b12 (Polymun, Cat. No. AB011), PGDM1400 and PGT151 (both kindly provided by Dr. Devon Sok and International AIDS Vaccine Initiative).

Techniques: Binding Assay, One-tailed Test, MANN-WHITNEY, Neutralization

Journal: Nature Communications

Article Title: Contemporary HIV-1 consensus Env with AI-assisted redesigned hypervariable loops promote antibody binding

doi: 10.1038/s41467-024-48139-x

Figure Lengend Snippet: Consensus Env sequences were expressed as gp140 glycoproteins. Comparison of the binding of A plasma pools from 13 cohorts of PLWH and B monoclonal antibodies to consensus B Env with unmodified (in red) and redesigned (in blue) HV loops. The number of plasma samples in each pool is shown in parenthesis in panel A . Binding of plasma samples ( n = 13) C and monoclonal antibodies ( n = 7, with PGDM1400 and MPER antibodies excluded) ( D ) to the subtype B, C and CRF01_AE consensus Env with unmodified HV loops (red) and redesigned HV loops (blue) is compared using paired, non-parametric one-tailed Wilcoxon signed-rank tests. MPER antibodies and PGDM1400 were not included in the statistical tests as their epitopes are not presented on the gp140 glycoproteins. E Neutralization sensitivity of the consensus Env with or without redesigned HV loops for subtypes B, C, and CRF01_AE measured for ten bnAbs that targeted the CD4 binding site (VRC01 and 3BNC117), V2-apex (PG9 and PGT145), glycan supersite (10-1074 and PGT128), MPER (10E8 and 4E10) and mannose-dependent (2G12) epitopes as well as the sCD4. The neutralization sensitivity is reported as IC50 values (μg/ml) with the most sensitive viruses colored in red and the most resistant in gray. Neutralization sensitivity (IC50 values) are shown for the unmodified and redesigned HV loops and displayed separately for each Env clade ( n = 10) ( F ) or each bnAb ( G ). The increase in sensitivity with the redesigned HV loops is tested for each clade with one-tailed exact Wilcoxon-Pratt signed-rank tests. Only the 01_AE_2010s.hv_redesigned.1 is shown in panels F and G as it has a shorter V1HV than 01_AE_2010s.hv_redesigned.2. Source data for all panels are provided in the Source Data files.

Article Snippet: 293 T cells were transfected with 1 ug of plasmid DNA encoding an HIV-1 env gene (PolyJet™ In Vitro DNA Transfection Reagent (SignaGen Laboratories, Cat. No. SL100688) in 6-well plates and harvested at 48 h. Cells were split into separate staining reactions for monoclonal antibodies 3BNC117 (Cat. No. ARP-12474), 10-1074 (Cat. No. ARP-12477), 10E8 (Cat. No. ARP-12294), 447-52D (Cat. No. ARP-4030, all from BEI resources), b12 (Polymun, Cat. No. AB011), PGDM1400 and PGT151 (both kindly provided by Dr. Devon Sok and International AIDS Vaccine Initiative).

Techniques: Comparison, Binding Assay, One-tailed Test, Neutralization