d7324 polyclonal sheep ab  (Biosynth Carbosynth)

Journal: bioRxiv

Article Title: Lenacapavir binding to immature Gag triggers the emergence of giant HIV-1 virions

doi: 10.1101/2025.07.16.665102

Figure Lengend Snippet: a , b , Infectivity of HIV-1 virions produced from pNL4-3-transfected ( a ) and pJR-FL-transfected ( b ) 293T/Vpr-HiBiT cells treated with LEN, assessed by Tat-driven firefly luciferase activity in TZM-bl cells. c , Infectivity of virions from pNL4-3-transfected 293T/Vpr-HiBiT cells measured in CEM-GFP cells by flow cytometry, based on GFP-positive cells. d , Infectivity of virions from pNL4-3-transfected 293T/Vpr-HiBiT cells measured in Jurkat cells by flow cytometry, based on intracellular p24-staining. e , f , Infectivity of HIV-1 virions produced from pNL4-3-infected Jurkat/Vpr-HiBiT cells ( e ) and pNL4-3-infected primary CD4 + T cells ( f ) treated with LEN, assessed by Tat-driven firefly luciferase activity in TZM-bl cells. Data are presented as mean ± s.d. from three independent experiments (biological triplicates where applicable) ( a – f ). g , Representative western blot showing HIV-1 gp120 incorporation in the presence or absence of LEN. h , Quantification of gp120 incorporation normalized to p24 levels, based on ImageJ analysis. Data are presented as mean ± s.d. from four independent experiments. i , HIV-1 fusion assessed using a BlaM-Vpr reporter assay and flow cytometric detection of CCF4 dye cleavage in the presence of LEN. Data are presented as mean ± s.d. from three independent experiments.

Article Snippet: HIV-1 Gag, Env, and FLAG-tagged proteins were detected by immunoblotting using the following primary antibodies: HIV-1 Immunoglobulin G (HIV-Ig: NIH AIDS reagent program), anti-HIV-1 gp120 antibody (Cat. No. D7324; Aalto Bio Reagents Ltd. Dublin, Ireland), Anti-DYKDDDDK tag monoclonal antibody (Cat. No. 012-22384; Fujifilm), and anti-p24 Gag monoclonal (#24-4: NIH AIDS Research and Reference Reagent Program).

Techniques: Infection, Produced, Transfection, Luciferase, Activity Assay, Flow Cytometry, Staining, Western Blot, Reporter Assay

Journal: PLOS Pathogens

Article Title: Assessing bnAb potency in the context of HIV-1 envelope conformational plasticity

doi: 10.1371/journal.ppat.1012825

Figure Lengend Snippet: A. Schematic illustrating the experimental sCD4-induced opening of cell surface expressed Env leading to exposure of the co-receptor binding site. For detection by flow-cytometry mAb 17b was used as a co-receptor mimic. B. Top panel: Opening of cell surface expressed Envs by increasing concentrations of sCD4 was monitored by staining with mAb 17b. Env mutants were divided into two groups showing enhanced 17b binding in absence of sCD4 (right) or not (left) compared to JR-CSF wt Env. The N332A mutant served as additional control. All titrations were performed once. Color code of mutant sensitivity as in . Middle panel: magnification of top panel data indicating data points for 17b staining of individual Env mutants. Bottom panel: The bar graph depicts area under the curve values derived from the normalized MFI curves of the top panel graphs. C. Spearman correlation between 17b neutralization sensitivity of JR-CSF wildtype and mutant viruses and 17b binding according to B. Mutations directly affecting 17b binding were not included (see also ). D. Alanine-substitutions leading to moderate (blue) or high (red) generalized neutralization sensitivity of the JR-CSF Env were mapped onto the trimeric closed prefusion structure of the closely related JR-FL Env ectodomain (PDB: 5FYK; V1V2: yellow, V3: orange, β20-β21: brown, gp120: light grey, gp41: dark grey). Glycans on neutralization sensitive positions N156, N262 and N301 are depicted. The N197 glycan is not shown as JR-FL naturally lacks this PNGS.

Article Snippet: Briefly, gp120 was captured by sheep anti-gp120 antibody (D7324, Aalto Bioreagents, Ireland) coated the day before to the surface of high-binding 384-well ELISA plates (corning).

Techniques: Binding Assay, Flow Cytometry, Staining, Mutagenesis, Control, Derivative Assay, Neutralization, Glycoproteomics