rabbit polyclonal anti cxcr4  (Cell Signaling Technology Inc)

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: bioRxiv

Article Title: ANALYSIS OF FACTORS THAT REGULATE HIV-1 FUSION IN REVERSE

doi: 10.1101/2025.03.10.642481

Figure Lengend Snippet: A. Cell-cell fusion assays involve 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 are seeded together onto coverslips, and scored for GFP+ syncytia two days later. B. HIV-1 Env expression constructs included one that produces the WT HIV-1 NL4-3 envelope protein (Env-WT: pMDG-NL43-Env-WT or SVIII-Env-WT) and one that produces 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 expresses 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( , )P2 (CXCR4-318-PH and CXCR4-309-PH).

Article Snippet: Primary antibodies employed 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, Expressing, Construct, Residue, Variant Assay

Journal: bioRxiv

Article Title: ANALYSIS OF FACTORS THAT REGULATE HIV-1 FUSION IN REVERSE

doi: 10.1101/2025.03.10.642481

Figure Lengend Snippet: Cell-cell fusion assays were performed 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 and B , C and D , and E and 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 cotransfected split GFP constructs plus the indicated Env or CD4 plus CXCR4 expression construct variants. Note that numbers of syncytia for Env-753 were approximately ten-fold 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, and that differences between the sets were highly significant (P<0.001).

Article Snippet: Primary antibodies employed 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: bioRxiv

Article Title: ANALYSIS OF FACTORS THAT REGULATE HIV-1 FUSION IN REVERSE

doi: 10.1101/2025.03.10.642481

Figure Lengend Snippet: (A) Target cells expressing different Env variants are infected with a βGal-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 βGal 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 bGal-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.

Article Snippet: Primary antibodies employed 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, Plasmid Preparation, Variant Assay, Binding Assay, Virus, Activity Assay, Derivative Assay, Comparison

Journal: bioRxiv

Article Title: ANALYSIS OF FACTORS THAT REGULATE HIV-1 FUSION IN REVERSE

doi: 10.1101/2025.03.10.642481

Figure Lengend Snippet: Cells expressing the 753Stop (A) or WT HIV-1 Env (B) were infected with βgal 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 βgal assays and βgal 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.

Article Snippet: Primary antibodies employed 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: bioRxiv

Article Title: ANALYSIS OF FACTORS THAT REGULATE HIV-1 FUSION IN REVERSE

doi: 10.1101/2025.03.10.642481

Figure Lengend Snippet: Jurkat T cells, HIV-1-infected J1.1 Jurkat cells, and J1.1 cells induced with tumor necrosis factor alpha (J1.1+TNFα) 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-βGal, 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 βGal 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)

Article Snippet: Primary antibodies employed 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: bioRxiv

Article Title: ANALYSIS OF FACTORS THAT REGULATE HIV-1 FUSION IN REVERSE

doi: 10.1101/2025.03.10.642481

Figure Lengend Snippet: 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 were scored via βGal 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 cotransfection of 293 cells with a GagPol expression vector (psPAX2), a βGal transducing vector (pLVX-puro-Xho-ATG-βGal), 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 and 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-βGal, 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 μM AME prior to infections. In panel C , TMEM refers to cotransfection 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, and significant differences were as follows: 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.

Article Snippet: Primary antibodies employed 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, Generated, Cotransfection, Expressing, Plasmid Preparation, Transfection, Produced, Virus, Knock-Out