goat anti dpp4  (R&D Systems)

Journal: npj Viruses

Article Title: Middle East respiratory syndrome coronavirus (MERS-CoV) internalization does not rely on DPP4 cytoplasmic tail signaling

doi: 10.1038/s44298-024-00080-y

Figure Lengend Snippet: a Representative brightfield fluorescence image and a corresponding graph showing a reduction in the percentage of infection of MERS-PV-GFP in Huh7 cells upon treatment with MERS-CoV spike polyclonal antibody, compared to the mock-treated group (b) and (c) Bar graph showing the percentage of neutralization of MERS-CoV PV infection in Huh7 cells and HEK293T cells transiently expressing DPP4, respectively d Relative percentage difference in MERS-CoV PV infection on HEK293T cells transiently expressing either pcDNA, DPP4 alone, TMPRSS2 alone or DPP4 + TMPRSS2 both (e – g) Percentage of relative MERS-CoV PV infection in Huh7, Vero and Calu3 respectively on treatment with 100 µM Camostat mesylate, N = 3, bar graphs represent mean ± SEM, ns non-significant, * p < 0.05; ** p < 0.01; ** * p < 0.001; **** p < 0.0001.

Article Snippet: Samples were loaded into a 10% SDS-PAGE gel, and Western blotting was performed using a goat anti-DPP4 primary antibody (R&D, AF1180, 1:1000) and secondary with a rabbit anti-goat HRP-conjugated antibody (Immunotag, ITSAH238, 1:5000).

Techniques: Fluorescence, Infection, Neutralization, Expressing

Journal: npj Viruses

Article Title: Middle East respiratory syndrome coronavirus (MERS-CoV) internalization does not rely on DPP4 cytoplasmic tail signaling

doi: 10.1038/s44298-024-00080-y

Figure Lengend Snippet: a Multiple sequence alignment of DPP4 in different MERS-CoV susceptible and non-susceptible species, marking the putative phosphorylation site at the third amino acid position (letter ‘P’ marked in red). b Schematic representation of different mutations introduced in the cytoplasmic tail of DPP4. c Histograms depicting surface expression of wtDPP4 and its other mutants using flow cytometry. The values in the histogram depicts the percentage of FITC-positive cells. d Percentage difference of different DPP4 mutants surface expression as compared to wtDPP4. e Schematic representation of coronavirus spike protein. f In vitro expression confirmation of recombinant MERS-CoV spike S1-Fc protein using immunocytochemistry, scale bar = 5 µm (g) Molecular mass confirmation of purified recombinant MERS-CoV spike S1-Fc protein through western blot. h Histograms depicting surface binding of MERS-CoV spike S1 on DPP4 and its other mutant expressing cells using flow cytometry, values in the histogram plots indicate the percentage of FITC positive cells. i Percentage difference of MERS-CoV spike S1-Fc protein binding on different DPP4 mutants as compared to wild-type DPP4, N = 3, bar graph represents mean ± SD, ** p < 0.01,*** p < 0.001.

Article Snippet: Samples were loaded into a 10% SDS-PAGE gel, and Western blotting was performed using a goat anti-DPP4 primary antibody (R&D, AF1180, 1:1000) and secondary with a rabbit anti-goat HRP-conjugated antibody (Immunotag, ITSAH238, 1:5000).

Techniques: Sequencing, Phospho-proteomics, Expressing, Flow Cytometry, In Vitro, Recombinant, Immunocytochemistry, Purification, Western Blot, Binding Assay, Mutagenesis, Protein Binding

Journal: npj Viruses

Article Title: Middle East respiratory syndrome coronavirus (MERS-CoV) internalization does not rely on DPP4 cytoplasmic tail signaling

doi: 10.1038/s44298-024-00080-y

Figure Lengend Snippet: HEK293T stable cell lines expressing either full-length DPP4 ((HEK293TwtDPP4) or cytoplasmic tail-deleted DPP4 (HEK293TΔcytDPP4) were used to assess. a Cell surface expression and MERS-CoV S1 binding (scale bar = 50 µm), b Expression levels of wtDPP4 and ΔcytDPP4 by Western blot, and (c) confirmation of the cytoplasmic tail deletion in ΔcytDPP4 using reverse transcription PCR (RT-PCR) with two distinct primer sets, “Set1” primers were designed to amplify the both full-length DPP4 and ΔcytDPP4 sequence, while “Set2” primers include a forward primer binding site located immediately downstream of the cytoplasmic tail region, allowing amplification only of ΔcytDPP4. d Dual staining of ΔcytDPP4 (red) and SARS-CoV-2 spike S1 protein (green) to visualize colocalization (yellow), white square indicates zoomed area, scale bar = 10 µm. e Dual staining of ΔcytDPP4 (red) and MERS-CoV spike S1 protein (green) to visualize colocalization (yellow), white square indicates zoomed area, scale bar = 10 µm, (f) and (g) Percentage of relative pseudovirus infection in HEK293T wtDPP4 and HEK293T ΔcytDPP4 stable cell lines and cells transiently expressing wtDPP4 or ΔcytDPP4 respectively, N = 3, bar graphs represent mean ± SD, ns non-significant, * p < 0.05; ** p < 0.01; *** p < 0.001.

Article Snippet: Samples were loaded into a 10% SDS-PAGE gel, and Western blotting was performed using a goat anti-DPP4 primary antibody (R&D, AF1180, 1:1000) and secondary with a rabbit anti-goat HRP-conjugated antibody (Immunotag, ITSAH238, 1:5000).

Techniques: Stable Transfection, Expressing, Binding Assay, Western Blot, Reverse Transcription, Reverse Transcription Polymerase Chain Reaction, Sequencing, Amplification, Staining, Infection