mouse anti hcar  (R&D Systems)

Journal: Oncolytic Virotherapy

Article Title: CXCL12 retargeting of an adenovirus vector to cancer cells using a bispecific adapter

doi: 10.2147/OV.S112107

Figure Lengend Snippet: Summary of surface antigen expression in cancer cell lines

Article Snippet: Subsequently, cells were incubated in the dark with either PBS (unstained) or different primary antibodies, including 1) PE-labeled mouse antihuman CXCR4 monoclonal antibody (BD Biosciences, San Jose, CA, USA), 2) PE-labeled mouse antihuman CXCR7 monoclonal antibody (BD Biosciences), 3) PE-labeled mouse IgG2a κ isotype control (BD Biosciences), 4) PE-labeled mouse antihuman hCAR monoclonal antibody (Santa Cruz Biotechnology, Santa Cruz, CA, USA), 5) PE-labeled mouse IgG1 κ isotype control (Abcam, Cambridge, MA, USA), 6) FITC-labeled mouse antihuman integrin α v β 3 monoclonal antibody (EMD Millipore), 7) FITC-labeled mouse antihuman integrin α v β 5 monoclonal antibody (EMD Millipore), and 8) FITC-labeled mouse IgG1 κ isotype control (BD Biosciences).

Techniques: Expressing

Journal: Nucleic Acids Research

Article Title: Molecular basis of crosstalk in nuclear receptors: heterodimerization between PXR and CAR and the implication in gene regulation

doi: 10.1093/nar/gkac133

Figure Lengend Snippet: Reduction of hPXR levels leads to an increase in hCAR-dependent CYP2B6 expression in human HepaRG cells. ( A , B ) HepaRG cells were transfected with non-targeting siRNA (siNT) or pooled siRNA targeting hPXR (siPXR) at 25 nM for 48 h then treated with DMSO or CITCO for an additional 24 h. RT-qPCR was performed for (A) hPXR or (B) CYP2B6 RNA. FC, fold change over siNT and DMSO treated cells. ( C ) CYP2B6 mRNA levels in parental wild-type (WT) cells compared to those in hPXR KO HepaRG cells treated with DMSO or 1 μM CITCO. FC, fold change over DMSO treated WT cells. ( D ) Western blot of CYP2B6 and hPXR proteins in WT and hPXR KO HepaRG cells. β-actin was used as the protein loading control. * P < 0.05; *** P < 0.0005.

Article Snippet: For immunoprecipitation of endogenous hCAR from HepaRG cells, 10 μl of mouse anti-hCAR antibody or 5 μl of immunoglobulin G (IgG) (Santa Cruz Biotech, cat. no. sc-2025) control was added to 50 μl of beads, respectively.

Techniques: Expressing, Transfection, Quantitative RT-PCR, Western Blot, Control

Journal: Nucleic Acids Research

Article Title: Molecular basis of crosstalk in nuclear receptors: heterodimerization between PXR and CAR and the implication in gene regulation

doi: 10.1093/nar/gkac133

Figure Lengend Snippet: The hPXR ligand-binding domain is sufficient for interaction with hCAR. ( A ) Schematics showing hPXR in full-length (hPXR FL) or truncated (hPXR LBD, hPXR DBD, hPXR.2) forms. hPXR.2 lacks 37 residues (174–210) within the LBD. ( B ) The hPXR LBD interacts with hCAR. HEK-293 cells were co-transfected with MYC-hCAR, along with FLAG vector control (Ctrl), full-length hPXR (FL), FLAG-hPXR LBD (LBD), or FLAG-hPXR DBD (DBD). Co-IP assays using anti-FLAG antibodies were performed at 48 h post-transfection, followed by immunoblot analysis with anti-FLAG and anti-MYC. ( C ) hPXR.2 interacts with hCAR. HEK-293 cells were co-transfected with GFP-hCAR (or GFP vector) and hPXR.2 isoform. A co-IP assay was performed at 48 h post transfection, followed by immunoblot analysis with anti-GFP and anti-hPXR. ( D ) The hPXR LBD interacts with the hCAR LBD in a mammalian two-hybrid assay. HEK-293 cells were transfected with VP16 AD–hPXR LBD, GAL4 DBD–hCAR LBD, or vector controls (VP16 AD and GAL4 DBD) as indicated. At 48 h post-transfection, pG5 luciferase activity resulting from hPXR LBD and hCAR LBD co-expression was measured (the relative luciferase activity was obtained by normalizing firefly luciferase to Renilla luciferase) and compared to that with hCAR LBD expression alone. FC, fold change over cells transfected with vector controls (VP16 AD and GAL4 DBD). ( E ) The hPXR LBD inhibits hCAR activity. HepG2 cells were transfected with the CYP2B6 -luc reporter in combination with hCAR (0.25 μg) with or without 0.25 μg of full-length hPXR (hPXR FL), hPXR LBD, or hPXR DBD as shown. At 48 h post-transfection, the CYP2B6 promoter activity resulting from hPXR and hCAR co-expression was measured and compared to that with hCAR alone. *** P < 0.0005; ns, not significant. Immunoglobulin heavy chain (IgG HC, 55 kDa) and light chain (IgG LC, 25 kDa) were indicated with an arrow in (B). FC, fold change over cells transfected with vector controls (VP16 AD and GAL4 DBD).

Article Snippet: For immunoprecipitation of endogenous hCAR from HepaRG cells, 10 μl of mouse anti-hCAR antibody or 5 μl of immunoglobulin G (IgG) (Santa Cruz Biotech, cat. no. sc-2025) control was added to 50 μl of beads, respectively.

Techniques: Ligand Binding Assay, Transfection, Plasmid Preparation, Control, Co-Immunoprecipitation Assay, Western Blot, Two Hybrid Assay, Luciferase, Activity Assay, Expressing

Journal: Journal of Virology

Article Title: Human Desmoglein-2 and Human CD46 Mediate Human Adenovirus Type 55 Infection, but Human Desmoglein-2 Plays the Major Roles

doi: 10.1128/JVI.00747-20

Figure Lengend Snippet: HAdV55 infection is positively correlated with the expression level of hDSG2. (A) Infection efficiency of HAdV55-EGFP in different cell lines derived from humans and monkeys. Each indicated cell line was infected with HAdV55-EGFP at 0, 100, 200, and 500 vp per cell. At 24 h postinfection, cells were examined using flow cytometry. The frequency of EGFP-positive cells is shown. Data are presented as the means ± SD. (B) Expression level of hDSG2 in different cell lines. (C) Expression level of hCD46 in different cell lines. (D) Expression level of hCAR in different cell lines. Each indicated cell line was stained with a FITC-labeled anti-hDSG2 antibody, a PE-labeled anti-hCD46 antibody, or a PE-labeled anti-hCAR antibody and examined using flow cytometry. The percentage of positive cells and the mean fluorescence intensity (MFI) of each cell line are shown. (E) Correlation of HAdV55 infectivity to the expression level of hDSG2. (F) Correlation of HAdV55 infectivity to the expression level of hCD46. (G) Correlation of HAdV55 infectivity to the expression level of hCAR. The infectivity of HAdV55 is presented as the frequency of EGFP-positive cells resulting from HAdV55-EGFP infection at 200 vp per cell. Representative results from three independent experiments are shown. The strength of the correlation was measured by the Pearson correlation coefficient.

Article Snippet: Mouse monoclonal antibodies to human β-actin, hDSG2 (6D8 and 8E5), or hCD46 (M177 and E4.3), phycoerythrin (PE)-labeled mouse anti-hCAR antibody (E1-1), fluorescein isothiocyanate (FITC)- or PE-labeled goat anti-mouse antibody, and mouse IgG1 isotype were purchased from Santa Cruz Biotechnology, Inc. A rabbit anti-hDSG2 antibody (EPR6768) was purchased from Abcam.

Techniques: Infection, Expressing, Derivative Assay, Flow Cytometry, Staining, Labeling, Fluorescence

Journal: Journal of Virology

Article Title: Human Desmoglein-2 and Human CD46 Mediate Human Adenovirus Type 55 Infection, but Human Desmoglein-2 Plays the Major Roles

doi: 10.1128/JVI.00747-20

Figure Lengend Snippet: Transient expression of hDSG2 promotes HAdV55 infection in rodent cells. (A) Plasmid-mediated expression of hDSG2 and hCD46. CHO-K1 cells were transfected with either pCDNA4-hDSG2, pCDNA4-hCD46, or both and the resultant expression of hDSG2 or hCD46 was examined by Western blot analysis. Cells transfected with pCDNA4-hCAR or pCDNA4TO were used as negative controls. GAPDH was also examined as an internal control. The blots are from different gels with equal volumes of each sample loaded. (B) Transient expression of hDSG2 facilitates HAdV55 infection in CHO-K1 cells. (C) Transient expression of hDSG2 facilitates HAdV14a infection in CHO-K1 cells. Transfected cells were infected with HAdV55-SEAP or HAdV14a-SEAP at 200 vp/cell, and the SEAP activity in the cultured media was measured 24 h after infection. (D) HAdV5-mediated expression of hDSG2 and hCD46. Hepa1-6 cells were preinfected with either HAdV5-hDSG2, HAdV5-hCD46, or both, and the resultant expression of hDSG2 or hCD46 was examined by Western blot analysis. Cells preinfected with HAdV5-hCAR or empty HAdV5 were used as negative controls. GAPDH was also examined as an internal control. The blots are from different gels with equal volumes of each sample loaded. (E) HAdV5-mediated expression of hDSG2 facilitates HAdV55 infection in Hepa1-6 cells. (F) HAdV5-mediated expression of hDSG2 facilitates HAdV14a infection in Hepa1-6 cells. Cells preinfected with HAdV5-hDSG2 or HAdV5-hCD46 were infected with HAdV55-SEAP or HAdV14a-SEAP at 200 vp/cell. The SEAP activity in the cultured media was measured 24 h after infection. Data are representative of those from three independent experiments and presented as the means ± SD. Statistical analysis was conducted by one-way ANOVA (n = 3). ***, P < 0.001.

Article Snippet: Mouse monoclonal antibodies to human β-actin, hDSG2 (6D8 and 8E5), or hCD46 (M177 and E4.3), phycoerythrin (PE)-labeled mouse anti-hCAR antibody (E1-1), fluorescein isothiocyanate (FITC)- or PE-labeled goat anti-mouse antibody, and mouse IgG1 isotype were purchased from Santa Cruz Biotechnology, Inc. A rabbit anti-hDSG2 antibody (EPR6768) was purchased from Abcam.

Techniques: Expressing, Infection, Plasmid Preparation, Transfection, Western Blot, Activity Assay, Cell Culture