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Jackson Immuno fab fragment
Fab Fragment, supplied by Jackson Immuno, used in various techniques. Bioz Stars score: 96/100, based on 465 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/result/fab fragment/product/Jackson Immuno
Average 96 stars, based on 465 article reviews

fab fragment - by Bioz Stars, 2026-05

96/100 stars

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Schematic illustration of PTR-SeNPs and MUC1@PTR-SeNPs synthesis and their anti-tumor efficacy against human triple-negative breast cancer.

Journal: Bioactive Materials

Article Title: Translational selenium nanoparticles trigger apoptosis in triple-negative breast cancer cells through the MAPKs/Bcl2 pathway

doi: 10.1016/j.bioactmat.2026.02.027

Figure Lengend Snippet: Schematic illustration of PTR-SeNPs and MUC1@PTR-SeNPs synthesis and their anti-tumor efficacy against human triple-negative breast cancer.

Article Snippet: Anti-human MUC1 therapeutic antibody Fab fragment (7B8) were purchased From Creative Biolabs (TAB-423MZ-F, USA).

Techniques:

Structure characterization of PTR-SeNPs and MUC1@ PTR-SeNPs. Structure characterization of PTR-SeNPs by (A) TEM, (B) Zetasizer Nano ZS, (C, D) Nanosight NS300, (E1-4) HRTEM-EDS and (F, G) FT-IR. (H) Confirmation of MUC1-C + PTR-SeNPs conjugation by confocal microscopy after fluorescent labeling with anti-mouse IgG (H + L). (I, J) Characterization results of the particle size and potential of MUC1@PTR-SeNPs

Journal: Bioactive Materials

Article Title: Translational selenium nanoparticles trigger apoptosis in triple-negative breast cancer cells through the MAPKs/Bcl2 pathway

doi: 10.1016/j.bioactmat.2026.02.027

Figure Lengend Snippet: Structure characterization of PTR-SeNPs and MUC1@ PTR-SeNPs. Structure characterization of PTR-SeNPs by (A) TEM, (B) Zetasizer Nano ZS, (C, D) Nanosight NS300, (E1-4) HRTEM-EDS and (F, G) FT-IR. (H) Confirmation of MUC1-C + PTR-SeNPs conjugation by confocal microscopy after fluorescent labeling with anti-mouse IgG (H + L). (I, J) Characterization results of the particle size and potential of MUC1@PTR-SeNPs

Article Snippet: Anti-human MUC1 therapeutic antibody Fab fragment (7B8) were purchased From Creative Biolabs (TAB-423MZ-F, USA).

Techniques: Conjugation Assay, Confocal Microscopy, Labeling

In vitro anti-tumor efficacy of PTR-SeNPs and MUC1@PTR-SeNPs on 17 TNBC c ell lines. ( A, B ) Protein expression level of MUC1 in 17 different TNBC cell lines. ( C, D ) IC 50 and maximum % growth inhibition of PTR-SeNPs and MUC1@PTR-SeNPs on 17 TNBC cell lines. ( E, G ) Cell cycle distribution triggered by PTR-SeNPs and MUC1@PTR-SeNPs in HCC1937 and MDA-MB-436 cells. After treatment with PTR-SeNPs or MUC1@PTR-SeNPs (4 and 40 μM) in HCC1937 and MDA-MB-436 cells for 72 h, cells were stained with propidium iodide followed by flow cytometry analysis using MultiCycle software. The apoptotic cell death was quantified by measuring the sub-G1 cell population. ( F, H ) Phosphatidylserine translocation mediated by PTR-SeNPs and MUC1@PTR-SeNPs in HCC1937 and MDA-MB-436 cells. After treatment with MUC1@PTR-SeNPs (4 and 40 μM) for 48 h, cells were co-stained with propidium iodide and Annexin-V-FITC followed by flow cytometry analysis [early apoptotic subset: Annexin V+/PI- (green); late apoptotic subset: Annexin V+/PT+ (red)].

Journal: Bioactive Materials

Article Title: Translational selenium nanoparticles trigger apoptosis in triple-negative breast cancer cells through the MAPKs/Bcl2 pathway

doi: 10.1016/j.bioactmat.2026.02.027

Figure Lengend Snippet: In vitro anti-tumor efficacy of PTR-SeNPs and MUC1@PTR-SeNPs on 17 TNBC c ell lines. ( A, B ) Protein expression level of MUC1 in 17 different TNBC cell lines. ( C, D ) IC 50 and maximum % growth inhibition of PTR-SeNPs and MUC1@PTR-SeNPs on 17 TNBC cell lines. ( E, G ) Cell cycle distribution triggered by PTR-SeNPs and MUC1@PTR-SeNPs in HCC1937 and MDA-MB-436 cells. After treatment with PTR-SeNPs or MUC1@PTR-SeNPs (4 and 40 μM) in HCC1937 and MDA-MB-436 cells for 72 h, cells were stained with propidium iodide followed by flow cytometry analysis using MultiCycle software. The apoptotic cell death was quantified by measuring the sub-G1 cell population. ( F, H ) Phosphatidylserine translocation mediated by PTR-SeNPs and MUC1@PTR-SeNPs in HCC1937 and MDA-MB-436 cells. After treatment with MUC1@PTR-SeNPs (4 and 40 μM) for 48 h, cells were co-stained with propidium iodide and Annexin-V-FITC followed by flow cytometry analysis [early apoptotic subset: Annexin V+/PI- (green); late apoptotic subset: Annexin V+/PT+ (red)].

Article Snippet: Anti-human MUC1 therapeutic antibody Fab fragment (7B8) were purchased From Creative Biolabs (TAB-423MZ-F, USA).

Techniques: In Vitro, Expressing, Inhibition, Staining, Flow Cytometry, Software, Translocation Assay

In vivo anti-tumor efficacy of MUC1@PTR- SeNPs. (A) MUC1 mRNA expression in normal tissue and primary breast cancer tumor using GEPIA database. ( B ) MUC1 expression in tumor tissues of MDA-MB-468-bearing mice in preliminary study. (C – E) Dose-dependent study of tumor inhibition effect of MUC1@PTR-SeNPs [75 (Low), 375 (Mid) & 750 μg (High) Se/kg BW/day] on BALB/c nude mice transplanted with MDA-MB-468 xenograft after oral administration for 30 days. PTR-SeNPs (High; 750 μg Se/kg BW/day) was used to investigate the possible improvement of in vivo anti-tumor efficacy by the MUC1@PTR-SeNPs. Quantitative analysis of Se content (μg/g) in (F) blood and (G) tumor tissue of experimental mice. (H) H&E, Ki67 and Tunnel fluorescence staining of tumor sections to detect apoptosis in vivo . (I) Western blot analysis of PARP, p-Bcl-2, Bax and C-caspase-9 protein expression in tumor sections. (J) In the serum of each group of tumor-bearing mice, the results of blood biochemistry-related indexes were analyzed.

Journal: Bioactive Materials

Article Title: Translational selenium nanoparticles trigger apoptosis in triple-negative breast cancer cells through the MAPKs/Bcl2 pathway

doi: 10.1016/j.bioactmat.2026.02.027

Figure Lengend Snippet: In vivo anti-tumor efficacy of MUC1@PTR- SeNPs. (A) MUC1 mRNA expression in normal tissue and primary breast cancer tumor using GEPIA database. ( B ) MUC1 expression in tumor tissues of MDA-MB-468-bearing mice in preliminary study. (C – E) Dose-dependent study of tumor inhibition effect of MUC1@PTR-SeNPs [75 (Low), 375 (Mid) & 750 μg (High) Se/kg BW/day] on BALB/c nude mice transplanted with MDA-MB-468 xenograft after oral administration for 30 days. PTR-SeNPs (High; 750 μg Se/kg BW/day) was used to investigate the possible improvement of in vivo anti-tumor efficacy by the MUC1@PTR-SeNPs. Quantitative analysis of Se content (μg/g) in (F) blood and (G) tumor tissue of experimental mice. (H) H&E, Ki67 and Tunnel fluorescence staining of tumor sections to detect apoptosis in vivo . (I) Western blot analysis of PARP, p-Bcl-2, Bax and C-caspase-9 protein expression in tumor sections. (J) In the serum of each group of tumor-bearing mice, the results of blood biochemistry-related indexes were analyzed.

Article Snippet: Anti-human MUC1 therapeutic antibody Fab fragment (7B8) were purchased From Creative Biolabs (TAB-423MZ-F, USA).

Techniques: In Vivo, Expressing, Inhibition, Fluorescence, Staining, Western Blot

( A ) Representative Coomassie blue-stained polyacrylamide gel of 5 μg of purified recombinant X4-gp120 and commercial gp120 (gp120), used as a control (n=3). ( B ) Representative western blot of different amounts (μg indicated) of recombinant X4-gp120 and commercial gp120 (5 μg), used as control, analyzed with an anti-gp120 mAb (n=3). ( C ) Flow cytometry of X4-gp120 binding to JKCD4 + CXCR4 + (JKCD4 + X4 + ) and JKCD4 + CXCR4 - (JKCD4 + X4 - ) cells analyzed with an anti-Histidine mAb. Mean MFI values (and SD) are shown (n=3; n.s.=not significant). ( D ) Flow cytometry of X4-gp120 binding on Daudi cells (CD4 - X4 + ), pretreated or not with soluble recombinant human CD4 (sCD4), analyzed with an anti-His mAb. As control, the binding of sCD4 in the absence of X4-gp120 is also shown. Mean MFI values (and SD) are shown (n=3; **p≤0.01). Statistical significance was determined by paired t-test for panel C, and by two-way ANOVA in panel B. Figure 1—figure supplement 1—source data 1. Original files for Coomassie blue-stained polyacrylamide gel for . Figure 1—figure supplement 1—source data 2. PDF file containing original Coomassie blue-stained polyacrylamide gel for . Generation of functional recombinant X4-gp120. Coomassie blue-stained polyacrylamide gel of 5 μg of purified recombinant X4-gp120 and commercial gp120 (gp120), used as a control. Original files for Coomassie blue-stained polyacrylamide gel displayed in . Figure 1—figure supplement 1—source data 3. Original files for western blot analysis for . Figure 1—figure supplement 1—source data 4. PDF file containing original western blot for . Original membrane corresponding to , panel B. Western blot of different amounts (μg indicated) of different batches of recombinant X4-gp120 and commercial gp120 (5 μg), used as control, analyzed with an anti-gp120 mAb. Batch#3 was selected for further assays. Original files for western blot analysis displayed in .

Journal: eLife

Article Title: HIV-1 envelope glycoprotein modulates CXCR4 clustering and dynamics on the T cell membrane

doi: 10.7554/eLife.110354

Figure Lengend Snippet: ( A ) Representative Coomassie blue-stained polyacrylamide gel of 5 μg of purified recombinant X4-gp120 and commercial gp120 (gp120), used as a control (n=3). ( B ) Representative western blot of different amounts (μg indicated) of recombinant X4-gp120 and commercial gp120 (5 μg), used as control, analyzed with an anti-gp120 mAb (n=3). ( C ) Flow cytometry of X4-gp120 binding to JKCD4 + CXCR4 + (JKCD4 + X4 + ) and JKCD4 + CXCR4 - (JKCD4 + X4 - ) cells analyzed with an anti-Histidine mAb. Mean MFI values (and SD) are shown (n=3; n.s.=not significant). ( D ) Flow cytometry of X4-gp120 binding on Daudi cells (CD4 - X4 + ), pretreated or not with soluble recombinant human CD4 (sCD4), analyzed with an anti-His mAb. As control, the binding of sCD4 in the absence of X4-gp120 is also shown. Mean MFI values (and SD) are shown (n=3; **p≤0.01). Statistical significance was determined by paired t-test for panel C, and by two-way ANOVA in panel B. Figure 1—figure supplement 1—source data 1. Original files for Coomassie blue-stained polyacrylamide gel for . Figure 1—figure supplement 1—source data 2. PDF file containing original Coomassie blue-stained polyacrylamide gel for . Generation of functional recombinant X4-gp120. Coomassie blue-stained polyacrylamide gel of 5 μg of purified recombinant X4-gp120 and commercial gp120 (gp120), used as a control. Original files for Coomassie blue-stained polyacrylamide gel displayed in . Figure 1—figure supplement 1—source data 3. Original files for western blot analysis for . Figure 1—figure supplement 1—source data 4. PDF file containing original western blot for . Original membrane corresponding to , panel B. Western blot of different amounts (μg indicated) of different batches of recombinant X4-gp120 and commercial gp120 (5 μg), used as control, analyzed with an anti-gp120 mAb. Batch#3 was selected for further assays. Original files for western blot analysis displayed in .

Article Snippet: The following antibodies were used: anti-human CXCR4 monoclonal antibody (mAb; clone 44717) and phycoerythrin-conjugated anti-human CXCR4 mAb (clone 12G5; both from R&D Systems, Minneapolis, MN); goat F(ab’)2 anti-mouse IgG-PE (Southern Biotech, Birmingham, AL); anti-human CD4 mAb (clone OKT4; Biolegend, San Diego, CA); anti-histidine mAb (clone AD1.1.10; R&D Systems); rabbit anti-gp120 IIIb Ab ( ); rabbit anti-Gag p24 HIV-1 mAb (R&D Systems); and anti-phospho-AKT mAb (S473; #4060), anti-phospho-ERK1,2 mAb (T202/Y204; #9191), and anti-phospho-Lck mAb (Y505; #2751; all from Cell Signaling Technology, Danvers, MA); anti-tubulin mAb conjugated with rhodamine (Bio-Rad, Hercules, CA); phalloidin-TRITC (#P1951, Sigma-Merck, St Louis, MO); anti-ICAM 3 mAb (clone HP2/19) kindly donated by Dr. Francisco Sánchez Madrid (Instituto Sanitario Hospital Universitario La Princesa); goat anti-mouse-AF488 Ab (Thermo Fisher Scientific); anti-human gp120 mAb Fab fragments (clone 2G12; Polymun Scientific, Vienna, Austria); anti-human IgG Fab fragments (Jackson ImmunoResearch, West Grove, PA) conjugated to Abberior STAR RED (Abberior GmbH, Gottingen, Germany), kindly donated by Dr. Jakub Chojnacki (Germans Trias i Pujol Research Institute (IGTP)); anti-p24 HIV-1 (clone 37G12; Polymun Scientific) conjugated with Abberior STAR ORANGE.

Techniques: Staining, Purification, Recombinant, Control, Western Blot, Flow Cytometry, Binding Assay, Functional Assay, Membrane

( A ) Western blot of lysed Jurkat cells activated with CXCL12 (50 nM) or X4-gp120 at the indicated time points and analyzed with anti-pAkt and pERK1/2 antibodies. A representative membrane is shown (n=3). ( B ) Western blot of primary CD4 + T blast lysates activated with X4-gp120 at the indicated time points and analyzed using anti-pLck and -pERK1/2 antibodies. A representative membrane is shown (n=3). In both cases, membranes were reblotted with an anti-tubulin mAb as a loading control. Molecular markers (kDa) are also shown. Figure 1—figure supplement 2—source data 1. Original files for western blot analysis for . Figure 1—figure supplement 2—source data 2. PDF file containing original western blot for . Original membrane corresponding to , panel A. Western blot of lysed Jurkat cells activated with CXCL12 (50 nM) or X4-gp120 Batch#3 (0.05; 0.1; 0.3 μg/ml) at the indicated time points and analyzed with anti-pAkt and pERK1/2 antibodies. Membranes were reblotted with an anti-tubulin mAb as a loading control. Molecular markers (kDa) are also shown. , panel A shows western blot of lysed Jurkat cells activated with CXCL12 (50 nM) and X4-gp120 Batch#3 0.3 μg/ml. Original files for western blot analysis displayed in . Figure 1—figure supplement 2—source data 3. Original files for western blot analysis for . Figure 1—figure supplement 2—source data 4. PDF file containing original western blot for . Original membrane corresponding to , panel B. Western blot of primary CD4 + T blast lysates activated with CXCL12 (50 nM) and X4-gp120 Batch#3 (0.3 μg/ml) at the indicated time points and analyzed using anti-pLck and pERK1/2 antibodies. Membranes were reblotted with an anti-tubulin mAb as a loading control. Molecular markers (kDa) are also shown. , panel B shows western blot of lysed Jurkat cells activated with X4-gp120 Batch#3 0.3 μg/ml. Original files for western blot analysis displayed in .

Journal: eLife

Article Title: HIV-1 envelope glycoprotein modulates CXCR4 clustering and dynamics on the T cell membrane

doi: 10.7554/eLife.110354

Figure Lengend Snippet: ( A ) Western blot of lysed Jurkat cells activated with CXCL12 (50 nM) or X4-gp120 at the indicated time points and analyzed with anti-pAkt and pERK1/2 antibodies. A representative membrane is shown (n=3). ( B ) Western blot of primary CD4 + T blast lysates activated with X4-gp120 at the indicated time points and analyzed using anti-pLck and -pERK1/2 antibodies. A representative membrane is shown (n=3). In both cases, membranes were reblotted with an anti-tubulin mAb as a loading control. Molecular markers (kDa) are also shown. Figure 1—figure supplement 2—source data 1. Original files for western blot analysis for . Figure 1—figure supplement 2—source data 2. PDF file containing original western blot for . Original membrane corresponding to , panel A. Western blot of lysed Jurkat cells activated with CXCL12 (50 nM) or X4-gp120 Batch#3 (0.05; 0.1; 0.3 μg/ml) at the indicated time points and analyzed with anti-pAkt and pERK1/2 antibodies. Membranes were reblotted with an anti-tubulin mAb as a loading control. Molecular markers (kDa) are also shown. , panel A shows western blot of lysed Jurkat cells activated with CXCL12 (50 nM) and X4-gp120 Batch#3 0.3 μg/ml. Original files for western blot analysis displayed in . Figure 1—figure supplement 2—source data 3. Original files for western blot analysis for . Figure 1—figure supplement 2—source data 4. PDF file containing original western blot for . Original membrane corresponding to , panel B. Western blot of primary CD4 + T blast lysates activated with CXCL12 (50 nM) and X4-gp120 Batch#3 (0.3 μg/ml) at the indicated time points and analyzed using anti-pLck and pERK1/2 antibodies. Membranes were reblotted with an anti-tubulin mAb as a loading control. Molecular markers (kDa) are also shown. , panel B shows western blot of lysed Jurkat cells activated with X4-gp120 Batch#3 0.3 μg/ml. Original files for western blot analysis displayed in .

Techniques: Western Blot, Membrane, Control

( A ) CD4+T cell blasts were perfused in flow chambers coated with ICAM-1-containing lipid bilayers, alone or with CXCL12- or X4-gp120. Polarized cells were determined from differential interference contrast (DIC) images using ImageJ2 and Imaris 7.0 and expressed as a percentage of the total number of cells per image (mean ± SD with the mean value of each experiment (black circles) indicated; n=6; n.s. not significant; *p≤0.05; ***p≤0.001). ( B ) Cells were perfused as in ( A ) and the percentage of adhered cells was determined using the interference reflection microscopy (IRM) signal. The frequency of adhesion (IRM + cells) per image field was estimated as [n° of cells showing IRM contact/total n° of cells (estimated by DIC)]×100. More than 150 cells were analyzed of each condition. Data are shown as percentage of adhered cells. (Mean ± SD with the mean value of each experiment (black circles) indicated; n=6; *p≤0.05; ***p≤0.001). ( C ) Cells in B were analyzed for cell migration. We calculated the frequency of migration (cells showing an IRM + contact and moving over time). Data are presented as percentage of migrating cells (mean ± SD with the mean value of each experiment (black circles) indicated; n=6; n.s.=not significant; **p≤0.01; *** p≤0.001). Statistical significance was determined by one-way ANOVA followed by Tukey’s multiple comparisons test for all panels.

Journal: eLife

Article Title: HIV-1 envelope glycoprotein modulates CXCR4 clustering and dynamics on the T cell membrane

doi: 10.7554/eLife.110354

Figure Lengend Snippet: ( A ) CD4+T cell blasts were perfused in flow chambers coated with ICAM-1-containing lipid bilayers, alone or with CXCL12- or X4-gp120. Polarized cells were determined from differential interference contrast (DIC) images using ImageJ2 and Imaris 7.0 and expressed as a percentage of the total number of cells per image (mean ± SD with the mean value of each experiment (black circles) indicated; n=6; n.s. not significant; *p≤0.05; ***p≤0.001). ( B ) Cells were perfused as in ( A ) and the percentage of adhered cells was determined using the interference reflection microscopy (IRM) signal. The frequency of adhesion (IRM + cells) per image field was estimated as [n° of cells showing IRM contact/total n° of cells (estimated by DIC)]×100. More than 150 cells were analyzed of each condition. Data are shown as percentage of adhered cells. (Mean ± SD with the mean value of each experiment (black circles) indicated; n=6; *p≤0.05; ***p≤0.001). ( C ) Cells in B were analyzed for cell migration. We calculated the frequency of migration (cells showing an IRM + contact and moving over time). Data are presented as percentage of migrating cells (mean ± SD with the mean value of each experiment (black circles) indicated; n=6; n.s.=not significant; **p≤0.01; *** p≤0.001). Statistical significance was determined by one-way ANOVA followed by Tukey’s multiple comparisons test for all panels.

Techniques: Microscopy, Migration

Single-particle tracking analysis of JKCD4 + X4 - cells transiently transfected with CXCR4-AcGFP on fibronectin (FN)-, FN +CXCL12-, or FN +X4-gp120-coated coverslips (828 particles in 96 cells on FN; 2997 in 95 cells on FN +CXCL12 and 1547 in 91 cells on FN +X4-gp120) n=3. ( A ) Percentage of mobile and immobile CXCR4-AcGFP particles at the membrane of cells treated as indicated. ( B ) Diffusion coefficients (D 1–4 ) of mobile particles at the membrane of cells treated as indicated with the median value of each experiment (black circles) and the median of all trajectories (dotted black lines; ****p≤0.0001). ( C ) Frequency of CXCR4-AcGFP particles containing monomers and dimers (≤2) or nanoclusters (≥3), mean ± SD calculated from mean spot intensity (MSI) values of each particle as compared with the value of monomeric CD86-AcGFP (980±86 a.u., **p≤0.01, ***p≤0.001). ( D ) Intensity distribution of individual CXCR4-AcGFP trajectories on unstimulated and CXCL12 or X4-gp120-stimulated cells. Graph shows the distribution of all trajectories, with the mean value of each experiment (black circles) and the median of all trajectories (dotted black lines; n=3; ****p≤0.0001). Statistical significance was determined by two-way ANOVA in panels A and C and by non-parametric Kruskal-Wallis tests followed by Dunn’s test for panels B and D .

Journal: eLife

Article Title: HIV-1 envelope glycoprotein modulates CXCR4 clustering and dynamics on the T cell membrane

doi: 10.7554/eLife.110354

Figure Lengend Snippet: Single-particle tracking analysis of JKCD4 + X4 - cells transiently transfected with CXCR4-AcGFP on fibronectin (FN)-, FN +CXCL12-, or FN +X4-gp120-coated coverslips (828 particles in 96 cells on FN; 2997 in 95 cells on FN +CXCL12 and 1547 in 91 cells on FN +X4-gp120) n=3. ( A ) Percentage of mobile and immobile CXCR4-AcGFP particles at the membrane of cells treated as indicated. ( B ) Diffusion coefficients (D 1–4 ) of mobile particles at the membrane of cells treated as indicated with the median value of each experiment (black circles) and the median of all trajectories (dotted black lines; ****p≤0.0001). ( C ) Frequency of CXCR4-AcGFP particles containing monomers and dimers (≤2) or nanoclusters (≥3), mean ± SD calculated from mean spot intensity (MSI) values of each particle as compared with the value of monomeric CD86-AcGFP (980±86 a.u., **p≤0.01, ***p≤0.001). ( D ) Intensity distribution of individual CXCR4-AcGFP trajectories on unstimulated and CXCL12 or X4-gp120-stimulated cells. Graph shows the distribution of all trajectories, with the mean value of each experiment (black circles) and the median of all trajectories (dotted black lines; n=3; ****p≤0.0001). Statistical significance was determined by two-way ANOVA in panels A and C and by non-parametric Kruskal-Wallis tests followed by Dunn’s test for panels B and D .

Techniques: Single-particle Tracking, Transfection, Membrane, Diffusion-based Assay

( A ) Representative transmission electron micrographs of gp120-VLP particles. Original scale bar 100 nm. ( B ) Culture media of transfected HEK-293T cells (cm) with different constructs, as indicated, and the corresponding clarified samples (cs) containing the VLPs or the LVPs generated, were analyzed by western blot with anti-p24, and -gp120 mAbs. The anti-p24 mAb also recognizes Pr55Gag, a precursor of p24 presents in immature particles. Molecular weight markers are indicated (kDa). ( C ) Flow cytometry analysis of VLPs (Env(-) and expressing x4-gp120) bound to latex beads in the presence of soluble human CD4, using an anti-Histidine mAb. A representative experiment is shown of 3 performed. ( D ) Representative transduction experiments using the indicated LVPs, where the reporter expression was captured using the Tecan SparkCyto reader. Upper panels show bright-field images of target HEK-293 CD4 cells. Lower panels show fluorescence signal from GFP expression in transduced cells. Images were captured with a 4× objective, using an exposure of 200ms in all cases and 80ms for control VSVG-VLPs (positive control). Env(-) LVPs were used as negative control (n=3). ( E ) Quantification of the Mean Fluorescence Intensity (MFI) of images obtained in transduction experiments. Figure 2—figure supplement 1—source data 1. Original files for western blot analysis for . Figure 2—figure supplement 1—source data 2. PDF file containing original western blots for . Original membranes corresponding to , panel B. Culture media of transfected HEK-293T cells (cm) with different constructs, as indicated, and the corresponding clarified samples (cs) containing the VLPs or the LVPs generated, were analyzed by western blot with anti-p24, and -gp120 mAbs. The anti-p24 mAb also recognizes Pr55Gag, a precursor of p24 presents in immature particles. Molecular weight markers are indicated (kDa). , panel B shows the last six lanes of these membranes reorganized to separate LVPs from VLPs. Original files for western blot analysis displayed in .

Journal: eLife

Article Title: HIV-1 envelope glycoprotein modulates CXCR4 clustering and dynamics on the T cell membrane

doi: 10.7554/eLife.110354

Figure Lengend Snippet: ( A ) Representative transmission electron micrographs of gp120-VLP particles. Original scale bar 100 nm. ( B ) Culture media of transfected HEK-293T cells (cm) with different constructs, as indicated, and the corresponding clarified samples (cs) containing the VLPs or the LVPs generated, were analyzed by western blot with anti-p24, and -gp120 mAbs. The anti-p24 mAb also recognizes Pr55Gag, a precursor of p24 presents in immature particles. Molecular weight markers are indicated (kDa). ( C ) Flow cytometry analysis of VLPs (Env(-) and expressing x4-gp120) bound to latex beads in the presence of soluble human CD4, using an anti-Histidine mAb. A representative experiment is shown of 3 performed. ( D ) Representative transduction experiments using the indicated LVPs, where the reporter expression was captured using the Tecan SparkCyto reader. Upper panels show bright-field images of target HEK-293 CD4 cells. Lower panels show fluorescence signal from GFP expression in transduced cells. Images were captured with a 4× objective, using an exposure of 200ms in all cases and 80ms for control VSVG-VLPs (positive control). Env(-) LVPs were used as negative control (n=3). ( E ) Quantification of the Mean Fluorescence Intensity (MFI) of images obtained in transduction experiments. Figure 2—figure supplement 1—source data 1. Original files for western blot analysis for . Figure 2—figure supplement 1—source data 2. PDF file containing original western blots for . Original membranes corresponding to , panel B. Culture media of transfected HEK-293T cells (cm) with different constructs, as indicated, and the corresponding clarified samples (cs) containing the VLPs or the LVPs generated, were analyzed by western blot with anti-p24, and -gp120 mAbs. The anti-p24 mAb also recognizes Pr55Gag, a precursor of p24 presents in immature particles. Molecular weight markers are indicated (kDa). , panel B shows the last six lanes of these membranes reorganized to separate LVPs from VLPs. Original files for western blot analysis displayed in .

Techniques: Transmission Assay, Transfection, Construct, Generated, Western Blot, Molecular Weight, Flow Cytometry, Expressing, Transduction, Fluorescence, Control, Positive Control, Negative Control

( A ) Representative images of clarified VLPs visualized by STED microscopy. Upper panels show images of the indicated VLPs stained for Gag p24 (blue) and gp120 (red). Lower panels show ×10 magnification of equivalent images. White arrows indicate mature VLPs (p24 condensation). ( B ) Percentage of mature VLPs, analyzed from the images in ( A ) using TrackAnalyzer in ImageJ, based on p24 intensity and aggregation level (mean ± SD; n=2; ****p≤0.0001; the significance indicated on immature VLPs bar shows the difference with all other conditions). ( C ) Percentage of VLPs expressing gp120 on their surface, as analyzed in ImageJ (mean ± SD; n=2; ***p≤0.001). ( D ) Distribution of gp120 mean fluorescence intensity. Each spot corresponds to the mean fluorescence intensity for each analyzed VLP in a.u. The black line represents the mean of all values (****p≤0.0001). ( E ) Frequency of gp120 intensity/particle. Statistical significance was determined by one-way-ANOVA followed by Tukey’s multiple comparisons test in panels B and C and by Mann-Whitney analysis for panel D.

Journal: eLife

Article Title: HIV-1 envelope glycoprotein modulates CXCR4 clustering and dynamics on the T cell membrane

doi: 10.7554/eLife.110354

Figure Lengend Snippet: ( A ) Representative images of clarified VLPs visualized by STED microscopy. Upper panels show images of the indicated VLPs stained for Gag p24 (blue) and gp120 (red). Lower panels show ×10 magnification of equivalent images. White arrows indicate mature VLPs (p24 condensation). ( B ) Percentage of mature VLPs, analyzed from the images in ( A ) using TrackAnalyzer in ImageJ, based on p24 intensity and aggregation level (mean ± SD; n=2; ****p≤0.0001; the significance indicated on immature VLPs bar shows the difference with all other conditions). ( C ) Percentage of VLPs expressing gp120 on their surface, as analyzed in ImageJ (mean ± SD; n=2; ***p≤0.001). ( D ) Distribution of gp120 mean fluorescence intensity. Each spot corresponds to the mean fluorescence intensity for each analyzed VLP in a.u. The black line represents the mean of all values (****p≤0.0001). ( E ) Frequency of gp120 intensity/particle. Statistical significance was determined by one-way-ANOVA followed by Tukey’s multiple comparisons test in panels B and C and by Mann-Whitney analysis for panel D.

Techniques: Microscopy, Staining, Expressing, Fluorescence, MANN-WHITNEY

Single-particle tracking analysis of JKCD4 + X4 - cells transiently transfected with CXCR4-AcGFP, on fibronectin (FN)-, FN +VLPs-, or FN +gp120 VLPs-coated coverslips (1087 particles in 159 cells on FN; 1400 in 153 cells on FN +VLPs and 1061 in 160 cells on FN +gp120 VLPs) n=6. ( A ) Diffusion coefficients (D 1–4 ) of mobile particles at the membrane of cells treated as indicated. Figure shows the mean value of each experiment (black circles) and the median of all trajectories (dotted black lines; n=6; ****p≤0.0001). ( B ) Frequency of CXCR4-AcGFP particles containing monomers and dimers (≤2) or nanoclusters (≥3) in cells treated as indicated. Mean ± SD calculated from mean spot intensity (MSI) values of each particle as compared with the value of monomeric CD86-AcGFP (980±86 a.u., **p≤0.05, **p≤0.01, ****p≤0.0001). ( C ) Intensity distribution (arbitrary units, a.u.) from individual CXCR4-AcGFP trajectories on cells treated as indicated. Graph shows the distribution of all trajectories, with the mean value of each experiment (black circles) and the median of all trajectories (dotted black lines; n=6; ****p≤0.0001). Statistical significance was determined by non-parametric Kruskal-Wallis tests followed by Dunn’s test for panels A and C , and by two-way ANOVA in panel B .

Journal: eLife

Article Title: HIV-1 envelope glycoprotein modulates CXCR4 clustering and dynamics on the T cell membrane

doi: 10.7554/eLife.110354

Figure Lengend Snippet: Single-particle tracking analysis of JKCD4 + X4 - cells transiently transfected with CXCR4-AcGFP, on fibronectin (FN)-, FN +VLPs-, or FN +gp120 VLPs-coated coverslips (1087 particles in 159 cells on FN; 1400 in 153 cells on FN +VLPs and 1061 in 160 cells on FN +gp120 VLPs) n=6. ( A ) Diffusion coefficients (D 1–4 ) of mobile particles at the membrane of cells treated as indicated. Figure shows the mean value of each experiment (black circles) and the median of all trajectories (dotted black lines; n=6; ****p≤0.0001). ( B ) Frequency of CXCR4-AcGFP particles containing monomers and dimers (≤2) or nanoclusters (≥3) in cells treated as indicated. Mean ± SD calculated from mean spot intensity (MSI) values of each particle as compared with the value of monomeric CD86-AcGFP (980±86 a.u., **p≤0.05, **p≤0.01, ****p≤0.0001). ( C ) Intensity distribution (arbitrary units, a.u.) from individual CXCR4-AcGFP trajectories on cells treated as indicated. Graph shows the distribution of all trajectories, with the mean value of each experiment (black circles) and the median of all trajectories (dotted black lines; n=6; ****p≤0.0001). Statistical significance was determined by non-parametric Kruskal-Wallis tests followed by Dunn’s test for panels A and C , and by two-way ANOVA in panel B .

Techniques: Single-particle Tracking, Transfection, Diffusion-based Assay, Membrane

Percentage of mobile receptor trajectories classified as confined, free, or directed for JK CD4 + X4 - cells transiently transfected with CXCR4-AcGFP in steady state or after Env(-) VLPs or gp120 VLPs stimulation. Statistical significance was determined by two-way ANOVA test.

Journal: eLife

Article Title: HIV-1 envelope glycoprotein modulates CXCR4 clustering and dynamics on the T cell membrane

doi: 10.7554/eLife.110354

Figure Lengend Snippet: Percentage of mobile receptor trajectories classified as confined, free, or directed for JK CD4 + X4 - cells transiently transfected with CXCR4-AcGFP in steady state or after Env(-) VLPs or gp120 VLPs stimulation. Statistical significance was determined by two-way ANOVA test.

Techniques: Transfection

Single-particle tracking analysis of JKCD4 + X4 - cells transiently transfected with CXCR4 R334X -AcGFP, on fibronectin (FN)-, FN +VLPs-, or FN +gp120 VLPs-coated coverslips (341 particles in 63 cells on FN; 610 in 54 cells on FN +VLPs and 707 in 63 cells on FN +gp120 VLPs) n=2. ( A ) Intensity distribution (arbitrary units, a.u.) from individual CXCR4 R334X -AcGFP trajectories on cells treated as indicated. Graph shows the distribution of all trajectories, with the mean value of each experiment (black circles) and the median of all trajectories ± SD (dotted black lines; n=2; ****p≤0.0001). ( B ) Diffusion coefficients (D 1–4 ) of mobile single particle trajectories at the membrane of cells treated as indicated. Figure shows the mean value of each experiment (black circles) and the median of all trajectories (dotted black lines; n=2; n.s. not significant, *p≤0.05, ****p≤0.0001). ( C ) Frequency of CXCR4 R334X -AcGFP particles containing monomers plus dimers (≤2) or nanoclusters (≥3), ± SD calculated from mean spot intensity values of each particle as compared with the value of monomeric CD86-AcGFP (*p≤0.05, ***p≤0.001). Statistical significance was determined by non-parametric Kruskal-Wallis tests followed by Dunn’s test for panels A and C, and by two-way ANOVA in panel B.

Journal: eLife

Article Title: HIV-1 envelope glycoprotein modulates CXCR4 clustering and dynamics on the T cell membrane

doi: 10.7554/eLife.110354

Figure Lengend Snippet: Single-particle tracking analysis of JKCD4 + X4 - cells transiently transfected with CXCR4 R334X -AcGFP, on fibronectin (FN)-, FN +VLPs-, or FN +gp120 VLPs-coated coverslips (341 particles in 63 cells on FN; 610 in 54 cells on FN +VLPs and 707 in 63 cells on FN +gp120 VLPs) n=2. ( A ) Intensity distribution (arbitrary units, a.u.) from individual CXCR4 R334X -AcGFP trajectories on cells treated as indicated. Graph shows the distribution of all trajectories, with the mean value of each experiment (black circles) and the median of all trajectories ± SD (dotted black lines; n=2; ****p≤0.0001). ( B ) Diffusion coefficients (D 1–4 ) of mobile single particle trajectories at the membrane of cells treated as indicated. Figure shows the mean value of each experiment (black circles) and the median of all trajectories (dotted black lines; n=2; n.s. not significant, *p≤0.05, ****p≤0.0001). ( C ) Frequency of CXCR4 R334X -AcGFP particles containing monomers plus dimers (≤2) or nanoclusters (≥3), ± SD calculated from mean spot intensity values of each particle as compared with the value of monomeric CD86-AcGFP (*p≤0.05, ***p≤0.001). Statistical significance was determined by non-parametric Kruskal-Wallis tests followed by Dunn’s test for panels A and C, and by two-way ANOVA in panel B.

Techniques: Single-particle Tracking, Transfection, Diffusion-based Assay, Single Particle, Membrane

Percentage of mobile receptor trajectories classified as confined, free, or directed for JK CD4 + X4 - cells transiently transfected with CXCR4 R334X -AcGFP in steady state or after Env(-) VLPs or gp120 VLPs stimulation. Statistical significance was determined by two-way ANOVA test.

Journal: eLife

Article Title: HIV-1 envelope glycoprotein modulates CXCR4 clustering and dynamics on the T cell membrane

doi: 10.7554/eLife.110354

Figure Lengend Snippet: Percentage of mobile receptor trajectories classified as confined, free, or directed for JK CD4 + X4 - cells transiently transfected with CXCR4 R334X -AcGFP in steady state or after Env(-) VLPs or gp120 VLPs stimulation. Statistical significance was determined by two-way ANOVA test.

Techniques: Transfection

FRET saturation curves generated using HEK-293T cells transiently transfected with a constant amount of CD4-CFP DNA (2 μg) and increasing amounts of ( A ) CXCR4-YFP (0.5–8.0 μg), ( B ) CXCR4 R334X -YFP (0.5–8.0 μg) or ( C ) 5HT 2B DNA (0.5–12 μg). K D and FRET max values were calculated using a nonlinear regression equation for a single binding-site model (n=2). ( D ) FRET efficiency in HEK-293 cells transiently transfected with CXCR4-YFP/ CD4-CFP (ratio 15:9), in the absence or presence of gp120 VLPs or Env(-) VLPs. Data shows FRET efficiency (arbitrary units, a.u.; mean ± SD; n=3; n.s. not significant, *p≤0.05, ***p≤0.001). ( E ) FRET efficiency in HEK-293 cells transiently transfected with CXCR4 R334X -YFP/CD4-CFP (ratio 15:9), in the absence or presence of gp120-VLPs or Env(-) VLPs. Data shows FRET efficiency (a.u.; mean ± SD; n=3; n.s. not significant, *p≤0.05, ***p≤0.001, ****p≤0.0001). Statistical significance was determined by unpaired t-test in panels D and E .

Journal: eLife

Article Title: HIV-1 envelope glycoprotein modulates CXCR4 clustering and dynamics on the T cell membrane

doi: 10.7554/eLife.110354

Figure Lengend Snippet: FRET saturation curves generated using HEK-293T cells transiently transfected with a constant amount of CD4-CFP DNA (2 μg) and increasing amounts of ( A ) CXCR4-YFP (0.5–8.0 μg), ( B ) CXCR4 R334X -YFP (0.5–8.0 μg) or ( C ) 5HT 2B DNA (0.5–12 μg). K D and FRET max values were calculated using a nonlinear regression equation for a single binding-site model (n=2). ( D ) FRET efficiency in HEK-293 cells transiently transfected with CXCR4-YFP/ CD4-CFP (ratio 15:9), in the absence or presence of gp120 VLPs or Env(-) VLPs. Data shows FRET efficiency (arbitrary units, a.u.; mean ± SD; n=3; n.s. not significant, *p≤0.05, ***p≤0.001). ( E ) FRET efficiency in HEK-293 cells transiently transfected with CXCR4 R334X -YFP/CD4-CFP (ratio 15:9), in the absence or presence of gp120-VLPs or Env(-) VLPs. Data shows FRET efficiency (a.u.; mean ± SD; n=3; n.s. not significant, *p≤0.05, ***p≤0.001, ****p≤0.0001). Statistical significance was determined by unpaired t-test in panels D and E .

Techniques: Generated, Transfection, Binding Assay

( A ) Surface receptor expression of CXCR4 (white dots) or CXCR4 R334X (black dots), after stimulation with CXCL12 (blue lines) or X4-gp120 (red lines). Results show mean ± SD of the percentage of receptor expression at the cell surface (n=3). ( B ) Surface receptor expression of CD4 in JK CD4 + CXCR4 + (white dots) or JK CD4 + CXCR4 R334X (black dots), after stimulation with CXCL12 (blue lines) or X4-gp120 (red lines). Results show mean ± SD of the percentage of receptor expression at the cell surface (n=3). Statistical significance was determined by one-way-ANOVA of AUC (*p≤0.05, **p≤0.01).

Journal: eLife

Article Title: HIV-1 envelope glycoprotein modulates CXCR4 clustering and dynamics on the T cell membrane

doi: 10.7554/eLife.110354

Figure Lengend Snippet: ( A ) Surface receptor expression of CXCR4 (white dots) or CXCR4 R334X (black dots), after stimulation with CXCL12 (blue lines) or X4-gp120 (red lines). Results show mean ± SD of the percentage of receptor expression at the cell surface (n=3). ( B ) Surface receptor expression of CD4 in JK CD4 + CXCR4 + (white dots) or JK CD4 + CXCR4 R334X (black dots), after stimulation with CXCL12 (blue lines) or X4-gp120 (red lines). Results show mean ± SD of the percentage of receptor expression at the cell surface (n=3). Statistical significance was determined by one-way-ANOVA of AUC (*p≤0.05, **p≤0.01).

Techniques: Expressing

The presence of CXCR4 R334X on JKCD4 + cells does not alter gp120 binding and increases fusion events with target cells expressing HIV pHXB2 envelope. ( A ) Binding of X4-gp120 to target cells expressing CD4 and CXCR4 or CD4 and CXCR4 R334X analyzed by flow cytometry. Cells were incubated with 0.3 mg/mL of X4-gp120 at 37 °C for 30 min. Data show MFI (arbitrary units, a.u.) mean ± SD; (n=2). Statistical significance was determined using Student’s t-test (n.s.=not significant). ( B ) Cell-cell fusion between JKHXBc2-expressing HIV-1 envelope and different target cells (JKCD4 + CXCR4 + , JKCD4 + CXCR4 - , and JKCD4 + CXCR4 R334X ). Prior to co-culture, each cell type was loaded with the corresponding cell-tracker. Data show the percentage of fusion events ± SD (n=6). We used as reference the fusions events detected in JKCD4 + CXCR4 + cells (100%). Statistical significance was determined by one-way-ANOVA (*p<0.05, ****p≤0.0001). ( C ) Representative biparametric histograms from cells in B showing CMAC versus orange fluorophores. ( D ) Human PBMCs isolated from a WHIM patient (WHIM) and three healthy donors (HD1-3) in two independent experiments were infected with X4-pseudotyped HIV-1 NL4-3 (MOI: 0.001). At 2 hr post infection (p.i.), supernatant samples were obtained at different time points (days post-infection) and p24 levels (pg/mL) in each sample were determined using a commercial ELISA. Results show mean ± SD (n=2).

Journal: eLife

Article Title: HIV-1 envelope glycoprotein modulates CXCR4 clustering and dynamics on the T cell membrane

doi: 10.7554/eLife.110354

Figure Lengend Snippet: The presence of CXCR4 R334X on JKCD4 + cells does not alter gp120 binding and increases fusion events with target cells expressing HIV pHXB2 envelope. ( A ) Binding of X4-gp120 to target cells expressing CD4 and CXCR4 or CD4 and CXCR4 R334X analyzed by flow cytometry. Cells were incubated with 0.3 mg/mL of X4-gp120 at 37 °C for 30 min. Data show MFI (arbitrary units, a.u.) mean ± SD; (n=2). Statistical significance was determined using Student’s t-test (n.s.=not significant). ( B ) Cell-cell fusion between JKHXBc2-expressing HIV-1 envelope and different target cells (JKCD4 + CXCR4 + , JKCD4 + CXCR4 - , and JKCD4 + CXCR4 R334X ). Prior to co-culture, each cell type was loaded with the corresponding cell-tracker. Data show the percentage of fusion events ± SD (n=6). We used as reference the fusions events detected in JKCD4 + CXCR4 + cells (100%). Statistical significance was determined by one-way-ANOVA (*p<0.05, ****p≤0.0001). ( C ) Representative biparametric histograms from cells in B showing CMAC versus orange fluorophores. ( D ) Human PBMCs isolated from a WHIM patient (WHIM) and three healthy donors (HD1-3) in two independent experiments were infected with X4-pseudotyped HIV-1 NL4-3 (MOI: 0.001). At 2 hr post infection (p.i.), supernatant samples were obtained at different time points (days post-infection) and p24 levels (pg/mL) in each sample were determined using a commercial ELISA. Results show mean ± SD (n=2).

Techniques: Binding Assay, Expressing, Flow Cytometry, Incubation, Co-Culture Assay, Isolation, Infection, Enzyme-linked Immunosorbent Assay

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