polyclonal rabbit anti cxcr4 antibody  (ProSci Incorporated)

Journal: Animal Bioscience

Article Title: Transcriptomic analysis identifies CXCL12 as a novel candidate gene for litter size in rabbits

doi: 10.5713/ab.24.0640

Figure Lengend Snippet: CXCL12 and its receptor CXCR4 were expressed and localized in the ovary, with their binding activating the JAK/STAT signaling pathway. (A) Immunofluorescence staining of ovarian follicles revealed that CXCL12 was co-localized with its receptor CXCR4 in ovarian follicles. (B) CXCL12 overexpression significantly promoted the expression of CXCR4 , JAK2 and STAT1 . (C) Knocking down CXCL12 significantly inhibited the expression of CXCR4 , JAK2 and STAT1 . (D) CXCL12 overexpression in GCs promoted the protein expression of CXCR4, JAK2 and STAT1 as well as the phosphorylation of JAK2 and STAT1. When CXCL12 was knocked down, the results were reversed. (E) MSX-122 inhibitor-based treatment further confirmed that CXCL12 promoted the total protein and phosphorylation levels of JAK2 and STAT1 (The t test was used for the above analyses comparing two individual samples. * p<0.05; ** p<0.01; *** p<0.001).

Article Snippet: These samples were then treated with 0.3% TritonX-100 (Beyotime) and 1% bovine serum albumin (Sigma, St. Louis, MO, USA) for 60 min prior to overnight incubation at 4°C with the following primary antibodies: anti-CXCL12 rabbit polyclonal antibody (1:250, Proteintech, Wuhan, China) and anti-CXCR4 rabbit polyclonal antibody (1:250, Proteintech).

Techniques: Binding Assay, Immunofluorescence, Staining, Over Expression, Expressing, Phospho-proteomics

Journal: Animal Bioscience

Article Title: Transcriptomic analysis identifies CXCL12 as a novel candidate gene for litter size in rabbits

doi: 10.5713/ab.24.0640

Figure Lengend Snippet: CXCL12 targets the receptor CXCR4 to activate the JAK/STAT signaling pathway and regulate the expression of related genes.

Article Snippet: These samples were then treated with 0.3% TritonX-100 (Beyotime) and 1% bovine serum albumin (Sigma, St. Louis, MO, USA) for 60 min prior to overnight incubation at 4°C with the following primary antibodies: anti-CXCL12 rabbit polyclonal antibody (1:250, Proteintech, Wuhan, China) and anti-CXCR4 rabbit polyclonal antibody (1:250, Proteintech).

Techniques: Expressing

Journal: Signal Transduction and Targeted Therapy

Article Title: Targeting TRPV6/CXCR4 complexes prevents castration-resistant prostate cancer metastasis to the bone

doi: 10.1038/s41392-025-02376-8

Figure Lengend Snippet: TRPV6 is directly involved in bone metastasis by coupling with the CXCR4 receptor. a Timeline of the experimental design of the bone metastasis model using Swiss-nude mice bearing either PC-3M-luc-C6 trpv6+/+ -mCherry or PC-3M-luc-C6 trpv6+/+ -pTRPV6 wt cell clones stably expressing firefly luciferase. I.C.: intracardiac. b Overall survival of PC-3M-luc-C6 trpv6+/+ -mCherry ( n = 9) versus PC-3M-luc-C6 trpv6+/+ -pTRPV6 wt ( n = 9) mice via the log rank (Mantel‒Cox) test. c Representative bioluminescence and X-ray images of mice prior to injection (left) and representative bioluminescence images and X-ray time latches of emerging bone metastases derived from PC-3M-luc-C6 trpv6+/+ -mCherry ( n = 9) versus PC-3M-luc-C6 trpv6+/+ -pTRPV6 wt ( n = 9) cell clones grafted via intracardiac injection. d Percentage of metastasis incidence in mice bearing PC-3M-luc-C6 trpv6+/+ -mCherry ( n = 9) versus PC-3M-luc-C6 trpv6+/+ -pTRPV6 wt ( n = 9) grafted cells. e Comparison of the metastasis-free survival of PC-3M-luc-C6 trpv6+/+ -mCherry ( n = 9) and PC-3M-luc-C6 trpv6+/+ -pTRPV6 wt ( n = 9) grafted mice via the log rank (Mantel‒Cox) test. f Representative X-ray image of mouse bone macrometastasis (black arrows) following intracardiac injection of PC-3M-luc-C6 trpv6+/+ -mCherry or PC-3M-luc-C6 trpv6+/+ -pTRPV6 wt cell clones. g Incidence of bone macrometastasis in mice grafted via intracardiac injections of either PC-3M-luc-C6 trpv6+/+ -mCherry ( n = 9) or PC-3M-luc-C6 trpv6+/+ -pTRPV6 wt ( n = 9) cell clones, as revealed via X-ray analysis or surgery. h Representative staining of TRPV6, CXCR4, and pankeratin as well as histological staining by H&E and Masson–Goldner’s trichrome in bone metastases (black triangles) from both the ribs and legs derived from intracardiac injections of either PC-3M-luc-C6 trpv6+/+ -mCherry or PC-3M-luc-C6 trpv6+/+ -pTRPV6 wt cell clones. The dotted rectangle represents the area used for zooming in on TRPV6 and CXCR4 staining. M: muscle. PCa: prostate cancer. B: bone. BM: bone marrow. A: Adipocytes. Black scale bars, 100 μm; red scale bars, 50 μm. i Quantification of both TRPV6- and CXCR4-positive cells involved in bone metastasis was performed following intracardiac injections of either PC-3 M-luc-C6 trpv6+/+ -mCherry or PC-3M-luc-C6 trpv6+/+ -pTRPV6 wt cell clones. j Membrane and total protein expression of CXCR4 in PC-3M trpv6−/− -mCherry, PC-3M trpv6−/− -pTRPV6 wt , and PC-3M trpv6−/− -pTRPV6 D582A stable cell clones. k Flow cytometric analysis of membrane CXCR4 expression in PC-3M trpv6−/− -mCherry, PC-3M trpv6−/− -pTRPV6 wt , and PC-3M trpv6−/− -pTRPV6 D582A stable cell clones. l Membrane and total protein expression of CXCR4 in PC-3M-luc-C6 trpv6+/+ -mCherry versus PC-3M-luc-C6 trpv6+/+ -pTRPV6 wt cell clones. m Flow cytometric analysis of membrane CXCR4 expression in PC-3M-luc-C6 trpv6+/+ -mCherry versus PC-3M-luc-C6 trpv6+/+ -pTRPV6 wt cell clones. n Membrane and total protein expression of CXCR4 in PC-3M-luc-C6 trpv6-/- versus PC-3M-luc-C6 trpv6+/+ cells. o Flow cytometric analysis of membrane CXCR4 expression in PC-3M-luc-C6 trpv6-/- versus PC-3M-luc-C6 trpv6+/+ cells. p Representative images and quantification (on the right) of TRPV6/CXCR4 coupling via the PLA in PC-3M trpv6−/− -mCherry, PC-3M trpv6−/− -pTRPV6 wt , and PC-3M trpv6−/− -pTRPV6 D582A stable cell clones; PC-3M-luc-C6 trpv6+/+ -mCherry versus PC-3M-luc-C6 trpv6+/+ -pTRPV6 wt cell clones; and PC-3M-luc-C6 trpv6−/− versus PC-3M-luc-C6 trpv6+/+ cells. Scale bars, 10 μm. q Directed migration and invasion of PC-3M-luc-C6 trpv6+/+ -mCherry versus PC-3M-luc-C6 trpv6+/+ -pTRPV6 wt cell clones treated with the specific CXCR4 inhibitor AMD3100 (30 μM) or vehicle to study the impact of CXCL12 as a chemoattractant (100 ng/mL). Representative images (left) and quantification of the number of cells that migrated and invaded through the matrix (right) ( n = 3). Scale bar, 200 μm. Mean ± SEM ( i , k , m , o–q ). Two-tailed t test ( d , g , i , m , o, p ). Two-way ANOVA ( k , q ). Log rank (Mantel‒Cox) test ( b , e ). See also Supplementary Fig.

Article Snippet: Then, the cells were resuspended in 3% PBS-BSA and stained with 15 μg/ml mouse monoclonal anti-TRPV6 antibody or 15 μg/ml rabbit polyclonal anti-CXCR4 antibody (clone D4Z7W, Cell Signaling Cat# 97680) for 2 h, followed by 3 washes.

Techniques: Clone Assay, Stable Transfection, Expressing, Luciferase, Injection, Derivative Assay, Comparison, Staining, Membrane, Migration, Two Tailed Test

Journal: Signal Transduction and Targeted Therapy

Article Title: Targeting TRPV6/CXCR4 complexes prevents castration-resistant prostate cancer metastasis to the bone

doi: 10.1038/s41392-025-02376-8

Figure Lengend Snippet: Effect of TRPV6 expression on bone metastasis development and bone architecture in vivo. a Timeline of the experimental bone-homing model using PC-3M-luc-C6 trpv6+/+ -mCherry and PC-3M-luc-C6 trpv6+/+ -pTRPV6 wt cell clones stably expressing firefly luciferase in Swiss-nude mice. I.O.: intraosseous. b Representative bioluminescence images and X-rays of mice bearing metastases from grafted i.o. PC-3M-luc-C6 trpv6+/+ -mCherry and PC-3M-luc-C6 trpv6+/+ -pTRPV6 wt cell clones. c Bone metastasis growth expressed as total photon flux in mice bearing intraosseous xenografts of PC-3M-luc-C6 trpv6+/+ -mCherry ( n = 10) and PC-3M-luc-C6 trpv6+/+ -pTRPV6 wt ( n = 10). d Representative images of Masson-Goldner’s trichrome, pankeratin, Ki67, TRPV6 and CXCR4 staining in tibias from mice bearing intraosseous xenografts of PC-3M-luc-C6 trpv6+/+ -mCherry and PC-3M-luc-C6 trpv6+/+ -pTRPV6 wt . Black scale bar, 2 mm. White scale bars, 20 μm. e and f 3D images of both the trabecular ( e ) and cortical ( f ) bone microarchitectures of the tibia and the BV/TV ratio, trabecular separation, trabecular number and trabecular thickness, cortical/total cross-sectional area, cortical thickness, total volume of pores, and total porosity expressed over the right tibia (control injection of PBS) of mice bearing intraosseous xenografts of PC-3M-luc-C6 trpv6+/+ -mCherry and PC-3M-luc- C6 trp6+/+ -pTRPV6 wt cell clones. g Timeline of the experimental bone-homing model using PC-3M-luc-C6 trpv6−/− and PC-3M-luc-C6 trpv6+/+ cell clones stably expressing firefly luciferase in Swiss-nude mice. I.O.: intraosseous. h Representative bioluminescence images and X-rays of mice bearing metastases derived from intraosseous xenografts of PC-3M-luc-C6 trpv6−/− and PC-3 M-luc-C6 trpv6+/+ cell clones. i Bone metastasis growth expressed as total photon flux in mice bearing intraosseous xenografts of PC-3M-luc-C6 trpv6−/− ( n = 10) and PC-3M-luc-C6 trpv6+/+ ( n = 10) mice. j Representative Masson–Goldner’s trichrome, pankeratin, Ki67, TRPV6 and CXCR4 staining of tibias from mice bearing intraosseous xenografts of PC-3M-luc-C6 trpv6−/− and PC-3M-luc-C6 trpv6+/+ cell clones. Black scale bars, 2 mm. White scale bars, 20 μm. k and l 3D images of both the trabecular ( k ) and cortical ( l ) bone microarchitectures at the tibia and the BV/TV ratio, trabecular separation, trabecular number and trabecular thickness, cortical/total cross-sectional area ratio, cortical thickness, total volume of pores, and total porosity expressed over the right tibia (control injection of PBS) of mice bearing intraosseous xenografts of PC-3M-luc-C6 trpv6−/− or PC-3M-luc-C6 trpv6+/+ cells. Mean ± SEM ( c , e , f , i , k , l ). Two-sided t test ( c , e , f , i , k , l ). See also Supplementary Fig.

Article Snippet: Then, the cells were resuspended in 3% PBS-BSA and stained with 15 μg/ml mouse monoclonal anti-TRPV6 antibody or 15 μg/ml rabbit polyclonal anti-CXCR4 antibody (clone D4Z7W, Cell Signaling Cat# 97680) for 2 h, followed by 3 washes.

Techniques: Expressing, In Vivo, Clone Assay, Stable Transfection, Luciferase, Staining, Control, Injection, Derivative Assay

Journal: Signal Transduction and Targeted Therapy

Article Title: Targeting TRPV6/CXCR4 complexes prevents castration-resistant prostate cancer metastasis to the bone

doi: 10.1038/s41392-025-02376-8

Figure Lengend Snippet: TRPV6 targeting via an anti-TRPV6 monoclonal antibody suppresses bone metastasis in vivo. a Timeline of the experimental bone metastasis model using Swiss mice grafted with PC-3M-luc-C6 trpv6+/ -pTRPV6 WT cell clones stably expressing firefly luciferase and treated with either 100 µg/kg mAbAU1 or mAb82 24 h before intracardiac cell injection, followed by treatment with either 100 µg/kg mAbAU1 or mAb82 twice per week starting 24 h after intracardiac cell injection. I.C.: intracardiac. b Overall survival of mice bearing PC-3M-luc-C6 trpv6+/+ -pTRPV6 wt cell clones and treated with either 100 µg/kg mAbAU1 ( n = 14) or mAb82 ( n = 14) 24 h after intracardiac cell injection, as determined via the log rank (Mantel‒Cox) test. c Representative bioluminescence images and X-rays of mice grafted with PC-3M-luc-C6 trpv6+/+ -pTRPV6 wt cell clones and treated with either 100 µg/kg mAbAU1 ( n = 14) or mAb82 ( n = 14) 24 h after intracardiac cell injection. d Incidence of bone metastasis in mice grafted with PC-3M-luc-C6 trpv6+/+ -pTRPV6 wt cell clones and treated with either 100 µg/kg mAbAU1 ( n = 14) or mAb82 ( n = 14) 48 h after intracardiac cell injection. e Survival of PC-3M-luc-C6 trpv6+/+ -pTRPV6 wt mice and mice treated with either 100 µg/kg mAbAU1 ( n = 14) or mAb82 ( n = 14) 24 h after intracardiac cell injection, as determined via the log rank (Mantel‒Cox) test. f Representative bioluminescence imaging and X-ray images of mice bearing metastases on the last day of treatment with either 100 µg/kg mAbAU1 or mAb82 (left) and representative H&E, Masson–Goldner’s trichrome and TRPV6 staining of bone metastases (right) from these mice. PCa: prostate cancer. B: bone. BM: bone marrow. Black scale bars, 100 µm. White scale bars, 20 µm. g Timeline of the experimental bone metastasis model using Swiss mice grafted with PC-3M-luc-C6 trpv6+/+ -pTRPV6 wt cell clones stably expressing firefly luciferase and treated with either 600 µg/kg mAbAU1 or mAb82 24 h before intracardiac cell injection, followed by treatment with either 100 µg/kg mAbAU1 or mAb82 twice per week starting 24 h after intracardiac cell injection. I.C.: intracardiac. h Overall survival of mice bearing PC-3 M-luc-C6 trpv6+/+ -pTRPV6 wt cell clones and treated with either 600 µg/kg mAbAU1 ( n = 12) or mAb82 ( n = 12) 24 h before intracardiac cell injection and then treated with either 100 µg/kg mAbAU1 or mAb82 twice per week starting 24 h after intracardiac cell injection, as determined via the log rank (Mantel‒Cox) test. i Representative bioluminescence imaging and X-ray images of mice grafted with PC-3M-luc-C6 trpv6+/+ -pTRPV6 wt cell clones and treated with either 600 µg/kg mAbAU1 ( n = 12) or mAb82 ( n = 12) 24 h before intracardiac cell injection, followed by treatment with either 100 µg/kg mAbAU1 or mAb82 twice per week starting 24 h after intracardiac cell injection. j Metastasis-free survival of mice bearing PC-3M-luc-C6 trpv6+/+ -pTRPV6 wt and treated with either 600 µg/kg mAbAU1 ( n = 12) or mAb82 ( n = 12) 24 h before intracardiac cell injection and then treated with either 100 µg/kg mAbAU1 or mAb82 twice per week starting 24 h after intracardiac cell injection, as determined via the log rank (Mantel‒Cox) test. k Incidence of bone metastasis in mice bearing PC-3M-luc-C6 trpv6+/+ -pTRPV6 wt and treated with either 600 µg/kg mAbAU1 ( n = 12) or mAb82 ( n = 12) 24 h before intracardiac cell injection, as determined via the log rank (Mantel‒Cox) test. l Representative H&E and Masson–Goldner’s Trichrome staining of bone metastases derived from the ribs of mice treated with either mAbAU1- or mAb82-treated mice as described in ( a ). PCa: prostate cancer. B: bone. BM: bone marrow. Scale bars, 100 µm. m Timeline of the experimental tumor growth model using PC-3M-luc-C6 trpv6+/+ cells stably expressing luciferase in Swiss-nude mice treated with either 100 µg/kg AU1 or mAb82 together with 5 mg/kg AMD3100 following tumor formation. S.C.: Subcutaneous. I.P.: Intraperitoneal. n Representative bioluminescence images of mice bearing xenografts of PC-3M-luc-C6 trpv6+/+ cells treated with either 100 µg/kg mAbAU1 or mAb82 together with 5 mg/kg AMD3100 following tumor formation. o Tumor growth in mice bearing xenografts of PC-3M-luc-C6 trpv6+/+ cells treated with either 100 µg/kg mAbAU1 or mAb82 together with 5 mg/kg AMD3100 following tumor formation. p Representative H&E, TRPV6, CXCR4, Ki-67 and TUNEL-fluorescein staining of primary tumors from mice bearing xenografts of PC-3M-luc-C6 trpv6+/+ cells treated with either 100 µg/kg mAbAU1 or mAb82 together with 5 mg/kg AMD3100 following tumor formation. Scale bar, 20 µm. q Survival assay (MTS) of PC-3M-luc-C6 trpv6+/+ cells treated with either 6 µg/mL mAbAU1/mAb82 together with 30 mM AMD3100 for 4 days. r Annexin V-FITC and IP analysis and quantification via flow cytometry of PC-3M-luc-C6 trpv6+/+ cells treated with either 6 µg/mL mAbAU1/mAb82 or 30 mM AMD3100 for 3 days. s Survival assay (MTS) of PC-3M-luc-C6 trpv6+/+ cells treated with either 6 µg/mL ulocuplumab or mAb82 or mAbAU1, as a control, for 4 days. t Annexin V-FITC and IP analysis and quantification via flow cytometry of PC-3M-luc-C6 trpv6+/+ cells treated with either 6 µg/mL ulocuplumab or mAb82 or mAbAU1, as a control, for 3 days. u Representative images and quantification of TRPV6/CXCR4 complexes revealed via the PLA in PC-3M-luc-C6 trpv6+/+ cells treated with either 6 µg/mL mAb82 together with 30 mM AMD3100 for 36 h. Scale bar, 10 µm. Mean ± SEM ( o , q– t ). Two-sided t test ( d , k ). Two-way ANOVA ( o , q–u ). Log rank (Mantel‒Cox) test ( b , e , h , k ). See also Supplementary Fig.

Article Snippet: Then, the cells were resuspended in 3% PBS-BSA and stained with 15 μg/ml mouse monoclonal anti-TRPV6 antibody or 15 μg/ml rabbit polyclonal anti-CXCR4 antibody (clone D4Z7W, Cell Signaling Cat# 97680) for 2 h, followed by 3 washes.

Techniques: In Vivo, Clone Assay, Stable Transfection, Expressing, Luciferase, Injection, Imaging, Staining, Derivative Assay, TUNEL Assay, Clonogenic Cell Survival Assay, Flow Cytometry, Control

Journal: Viruses

Article Title: Analysis of Factors That Regulate HIV-1 Fusion in Reverse

doi: 10.3390/v17040472

Figure Lengend Snippet: Cell–cell fusion assays and recombinant envelope and receptor constructs. ( A ). Cell–cell fusion assays involved transfection of one set of cells with an Env expression construct (in red) plus a PM-targeted split GFP helix 1–10 expression construct (pcDNA3.1-PLCdeltaPH-GFP-H1-10; green), and transfection of a separate set of cells with expression vectors for CD4 (blue) and CXCR4 (pink) variants, plus a PM-targeted split GFP helix 11 expression construct (For pGFP-H11-PLCdeltaPH; green). One day post-transfection cells were seeded together onto coverslips, and scored for GFP+ syncytia two days later. ( B ). HIV-1 Env expression constructs included one that produced the WT HIV-1 NL4-3 envelope protein (Env-WT: pMDG-NL43-Env-WT or SVIII-Env-WT) and one that produced an Env protein (Env-753stop: pMDG-NL43-Env-753stop), truncated at Env residue 753, before the CT amphipathic helices. CD4 expression constructs included one that expressed a Myc-tagged WT human CD4 protein (CD4: pCAGGS-CD4-Myc) or a variant in which the CD4 transmembrane and cytoplasmic tail (TMCT) domains were replaced with a GPI anchor (CD4-GPI: pCAGGS-CD4-GPI). CXCR4 expression constructs included a WT human CXCR4 expression construct (CXCR4: pcDNA3-CXCR4), constructs with partial (CXCR4-318: pcDNA3-CXCR4-318Stop) or complete (CXCR4-309: pcDNA3-CXCR4-309Stop) CXCR4 cytoplasmic tail (CT) deletions, and constructs in which CT truncations were fused to the PLCδPH that preferentially binds to PI(4,5)P2 (CXCR4-318-PH and CXCR4-309-PH).

Article Snippet: The employed primary antibodies were as follows: mouse anti-HIV-CA hybridoma media (Hy183, kindly provided by Dr. Bruce Chesebro) at 1:15; mouse anti-gp41(CT) hybridoma media (Chessie 8, recognizing CT residues 727-732 [PDRPEG], from the NIH AIDS Reagent Program) at 1:15; rabbit anti-CD4 D2E6M monoclonal antibody (Cell Signaling Technology, 93518T), used at 1:2000; polyclonal rabbit anti-CXCR4 antibody (ProSci, #1009), used at 1:2000.

Techniques: Recombinant, Construct, Transfection, Expressing, Produced, Residue, Variant Assay

Journal: Viruses

Article Title: Analysis of Factors That Regulate HIV-1 Fusion in Reverse

doi: 10.3390/v17040472

Figure Lengend Snippet: HIV-1 Env cell–cell fusion assays. Cell–cell fusion assays were performed with 293 cells as illustrated in . Panels ( A – F ) show coincubations of cells transfected with pGFP-H11-PLCdeltaPH plus CD4-GPI plus CXCR4-318 mixed with cells transfected with pcDNA3.1-PLCdeltaPH-GFP-H1-10 plus either Env-WT ( A , B ), Env-753Stop ( C , D ), or a mock ( E , F ). Panels ( A – F ) are matched photos imaged for DAPI nuclear stain ( A , C , E ) and GFP ( B , D , F ). Note that the size bar (panel ( F )) pertains to all fluorescent images ( A – F ). In panel ( G ), numbers of syncytia per 0.4 mm 2 are averaged with standard deviations from five images each of coincubations of cells co-transfected with split GFP constructs, plus the indicated Env or CD4 plus CXCR4 expression construct variants. Note that numbers of syncytia for Env-753 were approximately tenfold higher than syncytia for Env-WT, and that syncytia for Env-WT were approximately twenty-fold higher than syncytia for the Env-minus (No Env) control. Differences between the sets were highly significant ( p < 0.001), and were obtained by conversion of means and standard deviations to Z values for probability calculations.

Article Snippet: The employed primary antibodies were as follows: mouse anti-HIV-CA hybridoma media (Hy183, kindly provided by Dr. Bruce Chesebro) at 1:15; mouse anti-gp41(CT) hybridoma media (Chessie 8, recognizing CT residues 727-732 [PDRPEG], from the NIH AIDS Reagent Program) at 1:15; rabbit anti-CD4 D2E6M monoclonal antibody (Cell Signaling Technology, 93518T), used at 1:2000; polyclonal rabbit anti-CXCR4 antibody (ProSci, #1009), used at 1:2000.

Techniques: Transfection, Staining, Construct, Expressing, Control

Journal: Viruses

Article Title: Analysis of Factors That Regulate HIV-1 Fusion in Reverse

doi: 10.3390/v17040472

Figure Lengend Snippet: Infection of Env-expressing cells via fusion in reverse. ( A ) Target 293 cells expressing different Env variants are infected with a β-gal-transducing, HIV-1-based, lentivirus vector pseudotyped with a GPI-anchored CD4 variant (CD4-GPI) plus a C-terminally-truncated CXCR4 protein (CXCR4-318). Receptor plus co-receptor binding to cellularly expressed Env activate the Env fusion machinery, resulting in virus infection. ( B ) Shown are β-gal activity-derived infectivities of cells expressing Env-753Stop, Env-WT, or no Env (mock), normalized to the Env-753Stop cells. Note that results are averaged from five independent experiments each, are plotted on a log scale graph to facilitate comparison, and that standard deviations for the Env-753Stop and mock cells were too small to be observed on the graph. ( C ) Cells expressing the indicated Env proteins were infected with CD4-GPI plus CXCR4-318 pseudotyped β-gal-transducing HIV-1 lentivirus vectors in the absence (753, WT) or presence (753 + T20, WT + T20) of HIV-1 Env fusion inhibitor T20. Results for pairs of infections are normalized to the infectivities of viruses in the absence of T20. Averages and standard deviations derive from three separate infections, and differences between 753 and WT and WT and mock were highly significant ( p < 0.001), as were differences between untreated and treated samples. Probabilities were obtained by conversion of means and standard deviations to Z values, which were used for calculations.

Article Snippet: The employed primary antibodies were as follows: mouse anti-HIV-CA hybridoma media (Hy183, kindly provided by Dr. Bruce Chesebro) at 1:15; mouse anti-gp41(CT) hybridoma media (Chessie 8, recognizing CT residues 727-732 [PDRPEG], from the NIH AIDS Reagent Program) at 1:15; rabbit anti-CD4 D2E6M monoclonal antibody (Cell Signaling Technology, 93518T), used at 1:2000; polyclonal rabbit anti-CXCR4 antibody (ProSci, #1009), used at 1:2000.

Techniques: Infection, Expressing, Plasmid Preparation, Variant Assay, Binding Assay, Virus, Activity Assay, Derivative Assay, Comparison

Journal: Viruses

Article Title: Analysis of Factors That Regulate HIV-1 Fusion in Reverse

doi: 10.3390/v17040472

Figure Lengend Snippet: Fusion in reverse infectivities for different Env, receptor, and co-receptor combinations. 293 cells expressing the 753Stop ( A ) or WT HIV-1 Env ( B ) were infected with equivalent volumes of β-gal-transducing HIV-1 particles carrying the indicated receptor plus co-receptor combinations (CD4, CD4-GPI [gpi],—[mock], CXCR4, 318 [CXCR4-318], 309 [CXCR4-309], 318PH [CXCR4-318-PH], 309PH [CXCR4-309-PH]). At 72 h post-infection, cells were subjected to β-gal assays and β-gal activities are plotted as infectivities relative to viruses carrying the CD4-GPI/CXCR4-318 receptor/co-receptor combinations. Averages and standard deviations derive from the following numbers of experiments: 753/gpi/318, 9; 753/gpi/-, 2; 753/-/318, 2; 753/CD4/325, 2; 753/gpi/CXCR4, 4; 753/gpi/309, 3; 753/gpi/318PH, 5; 753/gpi/309PH, 5; WT/gpi/318, 9; WT/gpi/-, 2; WT/-/318, 2; WT/CD4/318, 3; WT/gpi/CXCR4, 6; WT/gpi/309, 4; WT/gpi/318PH, 5; WT/gpi/309PH, 5. Note that differences between the gpi/318 samples versus the gpi/-, -/318, and CD4/318 samples had probabilities of p < 0.001, as determined by conversion of means and standard deviations to Z values for probability calculations.

Article Snippet: The employed primary antibodies were as follows: mouse anti-HIV-CA hybridoma media (Hy183, kindly provided by Dr. Bruce Chesebro) at 1:15; mouse anti-gp41(CT) hybridoma media (Chessie 8, recognizing CT residues 727-732 [PDRPEG], from the NIH AIDS Reagent Program) at 1:15; rabbit anti-CD4 D2E6M monoclonal antibody (Cell Signaling Technology, 93518T), used at 1:2000; polyclonal rabbit anti-CXCR4 antibody (ProSci, #1009), used at 1:2000.

Techniques: Expressing, Infection

Journal: Viruses

Article Title: Analysis of Factors That Regulate HIV-1 Fusion in Reverse

doi: 10.3390/v17040472

Figure Lengend Snippet: Infection of HIV-1-infected cells. Jurkat T cells, HIV-1-infected J1.1 Jurkat cells, and J1.1 cells induced with tumor necrosis factor alpha (J1.1 + TNFa) were subjected to immunoblot detection of Gag proteins ( A ) with an anti-CA antibody, immunoblot detection of Env proteins ( B ) with an anti-gp41(TM) antibody, and infection with a lentivirus vector ( C ) generated by transfection of 293 cells with psPAX2, pLVX-puro-Xho-ATG-β-gal, CD4-GPI, and CXCR4-328 plasmids. ( A , B ) Migration positions of PrGag, CA, gp160 and gp41 proteins are indicated, as are molecular weight standards (in kDa) that were electrophoresced in parallel. ( C ) Infectivity results represent normalized transduced β-gal activity readings normalized to results for induced J1.1 cells. Averages and standard deviations derive from duplicate (Jurkat, J1.1) or triplicate (J1.1 + TNFα) infections, and differences between Jurkat and J1.1, as well as J1.1 versus J1.1 + TNFα were highly significant ( p < 0.001), as determined by conversion of means and standard deviations to Z values that were used in probability calculations.

Article Snippet: The employed primary antibodies were as follows: mouse anti-HIV-CA hybridoma media (Hy183, kindly provided by Dr. Bruce Chesebro) at 1:15; mouse anti-gp41(CT) hybridoma media (Chessie 8, recognizing CT residues 727-732 [PDRPEG], from the NIH AIDS Reagent Program) at 1:15; rabbit anti-CD4 D2E6M monoclonal antibody (Cell Signaling Technology, 93518T), used at 1:2000; polyclonal rabbit anti-CXCR4 antibody (ProSci, #1009), used at 1:2000.

Techniques: Infection, Western Blot, Plasmid Preparation, Generated, Transfection, Migration, Molecular Weight, Activity Assay

Journal: Viruses

Article Title: Analysis of Factors That Regulate HIV-1 Fusion in Reverse

doi: 10.3390/v17040472

Figure Lengend Snippet: Effects of lipid variations on fusion in reverse infections. ( A ) The HIV-1 viral membrane is highly enriched in cholesterol (CHOL), and has substantial amounts of ceramide (CER). The inner leaflets are enriched for phosphatidylserine (PS), phosphatidylinositol-4,5-bisphosphate (PI[4,5]P2; PIP2), and phosphatidylethanolamine (PE), while the outer leaflet is enriched in sphingomyelin (SM), hexosylceramides (HEX), and saturated phosphatidylcholines (PCs). ( B – D ) Infectivities of equivalent volumes of virus were scored via β-gal activities, and were normalized either to untreated (−) samples, or for convenience in the panel ( D ) CD/CX infections, to viruses from CerS−/− cells. In all panels, CD/CX viruses refer to lentivirus vectors generated by co-transfection of 293 cells with a GagPol expression vector (psPAX2), a β-gal-transducing vector (pLVX-puro-Xho-ATG-β-gal), as well as CD4-GPI and CXCR4-318 plasmids. Also, in all panels, Env-WT and Env-753 targets cells refer to 293 cells transfected to express those proteins. For panels ( B , C ), WT and 753 viruses are the NL4-3 variants and were used to infect TZM-bl reporter cells; for panel ( D ), WT refers to viruses produced from cells transfected with psPAX2, pLVX-puro-Xho-ATG-β-gal, plus a WT Env expression vector, while targets were 293 cells transfected to express the CD4-GPI and CXCR4-318 proteins. In panel ( B ), AME refers to treatment of viruses with 10 mM AME prior to infections. In panel ( C ), TMEM refers to co-transfection of virus producing cells with (+) or without (−) the mTMEM-GY scramblase expression vector. In panel ( D ), CerS2−/− pertains to producing viruses either from 293 cells (−) or from 293-CerS2−/− knockout cells (+). For panels ( B – D ), averages and standard deviations are as indicated, noting that where error bars are missing, it is because the standard deviations were too small to be depicted. The following significant differences were obtained by conversion of means and standard deviations to Z values for probability calculations: Panel ( B ) WT/TZMBL, AME −/+, p < 0.001; Panel ( B ) CDCX/Env-WT, AME −/+, p < 0.001; Panel C all pairs p < 0.001; Panel ( D ) CDCX/Env-WT, CerS2−/−, −/+, p < 0.01; all other Panel D pairs, p < 0.001. Note that in panel ( D ), if normalizations were based on CDCX viruses produced from WT 293 cells and infections of Env-753Stop-expressing cells, infectivities of CDCX viruses from virus–cell combinations would be as follows: from WT 293 cells into Env-753Stop cells, 100%; from CerS2−/− cells into Env-753Stop cells 500%; from WT 293 cells into Env-WT cells, 8%; from CerS2−/− cells into Env-WT cells, 19%.

Article Snippet: The employed primary antibodies were as follows: mouse anti-HIV-CA hybridoma media (Hy183, kindly provided by Dr. Bruce Chesebro) at 1:15; mouse anti-gp41(CT) hybridoma media (Chessie 8, recognizing CT residues 727-732 [PDRPEG], from the NIH AIDS Reagent Program) at 1:15; rabbit anti-CD4 D2E6M monoclonal antibody (Cell Signaling Technology, 93518T), used at 1:2000; polyclonal rabbit anti-CXCR4 antibody (ProSci, #1009), used at 1:2000.

Techniques: Membrane, Virus, Generated, Cotransfection, Expressing, Plasmid Preparation, Transfection, Produced, Knock-Out