goat polyclonal anti human dpp4 antibody  (R&D Systems)

Journal: Journal of Virology

Article Title: Comparison of Experimental Middle East Respiratory Syndrome Coronavirus Infection Acquired by Three Individual Routes of Infection in the Common Marmoset

doi: 10.1128/jvi.01739-21

Figure Lengend Snippet: Viral replication of MERS-CoV in BHK cells following transfection with either human or marmoset DPP4 receptor RNA. Significant differences were determined by ANOVA, with differences shown between mock-transfected cells (Lipofectamine) and human and marmoset transfected cells (**, P < 0.01; ***, P < 0.001).

Article Snippet: For DPP4 IHC staining, the tissue sections were subjected to heat-induced epitope retrieval using ER1, a citrate-based buffer (catalog number AR9961; Leica Biosystems), for 20 min at 95°C before applying a goat polyclonal anti-human DPP4 antibody (R&D Systems) diluted 1:250 and incubated for 15 min. A rabbit anti-goat secondary IgG antibody (Abcam, UK) was then applied for 8 min before using the Leica Intense R detection kit (Leica Biosystems) for visualization.

Techniques: Transfection

Journal: Journal of Virology

Article Title: Comparison of Experimental Middle East Respiratory Syndrome Coronavirus Infection Acquired by Three Individual Routes of Infection in the Common Marmoset

doi: 10.1128/jvi.01739-21

Figure Lengend Snippet: Location and activity of the DPP4 receptor in marmosets. (A and B) Immunohistochemical staining indicates a very strong presence of the DPP4 receptor within the alveolar spaces of marmosets (A) and no expression in the nasal cavity epithelium and moderate expression in the NALT and other submucosal structures (B). (C) The virus is observed in areas of high DPP4 receptor expression following aerosol challenge with MERS-CoV strain EMC/2012 within the terminal bronchioles and alveolar spaces. (D) An aggregate of MERS-CoV-positive lymphoid cells was observed in the nasal cavity. (E) Locations of MERS-CoV antigen and levels of expression of the DPP4 receptor in the respiratory tract and lungs of marmosets.

Article Snippet: For DPP4 IHC staining, the tissue sections were subjected to heat-induced epitope retrieval using ER1, a citrate-based buffer (catalog number AR9961; Leica Biosystems), for 20 min at 95°C before applying a goat polyclonal anti-human DPP4 antibody (R&D Systems) diluted 1:250 and incubated for 15 min. A rabbit anti-goat secondary IgG antibody (Abcam, UK) was then applied for 8 min before using the Leica Intense R detection kit (Leica Biosystems) for visualization.

Techniques: Activity Assay, Immunohistochemical staining, Staining, Expressing, Virus, Aerosol

Journal: Scientific Reports

Article Title: Insights into replicative senescence of human testicular peritubular cells

doi: 10.1038/s41598-019-51380-w

Figure Lengend Snippet: Proteomic analysis of advanced compared to early passages of HTPCs. Gene set enrichment analysis (GSEA) revealed significantly enriched gene sets (FDR q-value ≤ 0.05) and were summarized using REVIGO by clustering semantically similar GO terms. Each of the 20 characteristic gene sets enriched in early ( a ) and advanced passages ( b ) of HTPCs are shown. Color-coding refers to the corresponding highest GO hierarchy level. The x-axis shows the enrichment significance resulting from the GSEA and is depicted as –log10 (FDR q-value). The number of quantified proteins per gene set is shown in brackets. Volcano plots of intracellular and extracellular proteins, which are more abundant in passaged HTPC cellular proteomes ( c ) and secretomes ( d ) are depicted as red dots and proteins less abundant are shown as blue dots, respectively. Selected proteins with significant difference in abundance are labeled. P-values were calculated by a paired two-sample t -test. DPP4 expression in testicular peritubular cells ( e ). Light micrographs of immunohistochemical staining of human testicular sections. DPP4 is detected in several peritubular cells and cells of the interstitial space. Right micrograph: detail of the DPP4 staining (framed area). The negative control is without staining.

Article Snippet: Primary polyclonal goat anti-human DPP4 antibody (1:40, R&D Systems, Minneapolis, MN, USA) was used.

Techniques: Labeling, Expressing, Immunohistochemical staining, Staining, Negative Control

Journal: The Journal of Biological Chemistry

Article Title: Middle East respiratory syndrome coronavirus and bat coronavirus HKU9 both can utilize GRP78 for attachment onto host cells

doi: 10.1074/jbc.RA118.001897

Figure Lengend Snippet: GRP78 is abundantly expressed on the cell surface of mammalian cells. Surface GRP78 expression was detected on mammalian cell lines with flow cytometry with no cell permeabilization. The immunostaining was performed for human lung cell lines ( A ), human extrapulmonary cell lines, human primary macrophages, and human primary T cells ( B ), as well as nonhuman cell lines ( C ). D, percentage of GRP78-positive cells quantified with DPP4 included for comparisons. E, MFI of GRP78 on the cell surface was quantified with isotype and DPP4 staining included as controls. F, sequence homology between human GRP78 and GRP78 in other mammals. Gates in A–C represented the percentage of GRP78-positive cells. Data in D and E represented mean and standard deviation from three independent experiments.

Article Snippet: Goat polyclonal anti-DPP4 at 5 μg/ml (R&D, AF1180) and rabbit IgG at 5 μg/ml (ThermoFisher Scientific, 31235) were included as controls.

Techniques: Expressing, Flow Cytometry, Immunostaining, Staining, Sequencing, Standard Deviation

Journal: The Journal of Biological Chemistry

Article Title: Middle East respiratory syndrome coronavirus and bat coronavirus HKU9 both can utilize GRP78 for attachment onto host cells

doi: 10.1074/jbc.RA118.001897

Figure Lengend Snippet: Co-expression of GRP78 and DPP4 in human tissues. Immunostaining of GRP78 and DPP4 was performed on paraffin slides of normal human tissues. GRP78 was labeled with a polyclonal rabbit anti-GRP78 antibody, and DPP4 was labeled with a polyclonal goat anti-DPP4 antibody. Cell nuclei were labeled with DAPI. The co-expression of GRP78 and DPP4 was detected in the bronchus ( A ), bronchiole ( B ), and alveolus ( C ). The co-localization of GRP78 and DPP4 was examined at a higher magnification in D . Images were acquired with a Carl Zeiss LSM 710 system. Bars, 50 μm for A–C. Bars, 5 μm for D .

Article Snippet: Goat polyclonal anti-DPP4 at 5 μg/ml (R&D, AF1180) and rabbit IgG at 5 μg/ml (ThermoFisher Scientific, 31235) were included as controls.

Techniques: Expressing, Immunostaining, Labeling

Journal: The Journal of Biological Chemistry

Article Title: Middle East respiratory syndrome coronavirus and bat coronavirus HKU9 both can utilize GRP78 for attachment onto host cells

doi: 10.1074/jbc.RA118.001897

Figure Lengend Snippet: GRP78 is involved in MERS-CoV entry. Pseudovirus antibody blocking assays were performed in Huh7 ( A ) and BEAS2B ( B ) cells. A titration of GRP78 or isotype control antibodies from 0 to 2.5 μg/ml was added and pre-incubated with Huh7 and BEAS2B cells for 1 h at 37 °C. MERS–S-pseudovirus or VSV-G–pseudovirus was subsequently added at a ratio of 100 LP per cell for 1 h. Luciferase activity was determined at 72 h post-inoculation and was normalized to that of the mock-treated cells. C, antibody blocking assay was performed in Huh7 cells using infectious MERS-CoV. Huh7 cells were pre-incubated with antibodies at the indicated concentration for 1 h at 37 °C. The cells were then challenged with MERS-CoV at 1 m.o.i. for 1 h at 37 °C in the presence of the antibodies. After 1 h, the cells were washed and harvested. MERS-CoV entry was assessed with qPCR, and the result was normalized to that of the mock-treated cells. D, Huh7 or BEAS2B cells were treated with 75 n m GRP78, DPP4, or scrambled siRNA for 2 consecutive days. The knockdown efficiency was evaluated with Western blottings. E, siRNA-treated Huh7 or BEAS2B cells were infected with MERS-CoV at 1 m.o.i. for 1 h at 37 °C. After 1 h, the cells were harvested, and virus entry was evaluated with qPCR analysis. The result was normalized to that of the scrambled siRNA-treated cells. siRNA-treated BEAS2B cells were infected with MERS-CoV at 0.1 m.o.i. for 1 h at 37 °C. The cell lysates ( F ) and supernatants ( G ) were harvested at 24 and 48 h post-infection. MERS-CoV replication was evaluated with qPCR analysis. H, siRNA-treated MDM or HFL was infected with MERS-CoV at 1 m.o.i. for 2 h at 37 °C. After 2 h, the cells were harvested, and virus entry was evaluated with qPCR analysis ( I ). The result was normalized to that of the scrambled siRNA-treated cells. siRNA-treated MDM or HFL was infected with MERS-CoV at 0.1 m.o.i. for 1 h at 37 °C. The cell lysates ( J ) and supernatants ( K ) were harvested at 24 h post-infection. MERS-CoV replication was evaluated with qPCR analysis. In all panels, data represented mean and S.D. from three independent experiments. Statistical analyses were carried out using Student's t test. Statistical significance was indicated by asterisks when p < 0.05. ns means not significant.

Article Snippet: Goat polyclonal anti-DPP4 at 5 μg/ml (R&D, AF1180) and rabbit IgG at 5 μg/ml (ThermoFisher Scientific, 31235) were included as controls.

Techniques: Blocking Assay, Titration, Incubation, Luciferase, Activity Assay, Antibody Blocking Assay, Concentration Assay, Western Blot, Infection

Journal: The Journal of Biological Chemistry

Article Title: Middle East respiratory syndrome coronavirus and bat coronavirus HKU9 both can utilize GRP78 for attachment onto host cells

doi: 10.1074/jbc.RA118.001897

Figure Lengend Snippet: GRP78 is up-regulated on the surface of MERS-CoV–infected cells. A, Huh7 cells were infected with MERS-CoV at 0.01 and 0.1 m.o.i. and were harvested for flow cytometry analysis at 24 h post-infection. B, percentage of MERS-CoV N–positive cells was quantified. C, in parallel, cell surface and total DPP4 and GRP78 among mock- or MERS-CoV–infected samples were analyzed by flow cytometry. D, percentage of DPP4-positive cells and GRP78-positive cells in mock- or MERS-CoV–infected samples were quantified. Total DPP4 and GRP78 staining was performed by first permeabilizing the cells with 0.1% Triton X-100, whereas the surface DPP4 and GRP78 staining was performed in the absence of cell permeabilization. The gate in A represented the percentage of MERS-CoV N–positive cells. The gates in C represented the percentage of DPP4- ( upper panels ) and GRP78 ( lower panels )-positive cells. Data represented mean and S.D. derived from three independent experiments. Statistical analyses were carried out using Student's t test. Statistical significance was indicated by asterisks when p < 0.05. ns means not significant.

Article Snippet: Goat polyclonal anti-DPP4 at 5 μg/ml (R&D, AF1180) and rabbit IgG at 5 μg/ml (ThermoFisher Scientific, 31235) were included as controls.

Techniques: Infection, Flow Cytometry, Staining, Derivative Assay

Journal: The Journal of Biological Chemistry

Article Title: Middle East respiratory syndrome coronavirus and bat coronavirus HKU9 both can utilize GRP78 for attachment onto host cells

doi: 10.1074/jbc.RA118.001897

Figure Lengend Snippet: Sialic acids and GRP78 act independently to facilitate the surface attachment of MERS-CoV. A, Huh7 cells were treated with neuraminidase from C. perfringens , with or without pre-incubation with the GRP78 polyclonal antibody. The cells were subsequently challenged with MERS–S-pseudovirus and assessed at 72 h post-infection for pseudovirus entry. B, RLK cells were treated with neuraminidase from Clostridium perfringens , with or without pre-incubation with the GRP78 polyclonal antibody. The cells were subsequently challenged with HKU9–S-pseudovirus and assessed at 72 h post infection for pseudovirus entry. Pseudovirus entry was quantified using a microplate reader as relative light units ( RLU ). Data represented mean and standard deviation derived from three independent experiments. Statistical analyses were carried out using Student's t test. Statistical significance was indicated by asterisk marks when p < 0.05.

Article Snippet: Goat polyclonal anti-DPP4 at 5 μg/ml (R&D, AF1180) and rabbit IgG at 5 μg/ml (ThermoFisher Scientific, 31235) were included as controls.

Techniques: Incubation, Infection, Standard Deviation, Derivative Assay