rabbit polyclonal anti-denv 2 ns3 antibody  (GeneTex)

Journal: BMC Research Notes

Article Title: Oroxylin A shows limited antiviral activity towards dengue virus

doi: 10.1186/s13104-022-06040-0

Figure Lengend Snippet: Effect of OA on DENV infection. BHK-21 cells were mock or DENV 2 infected before being treated with an equivalent volume of DMEM (DM), or DMSO solvent control (DS) or various concentrations of OA for 24 h. A After 24 h levels of DENV E, NS1, NS3 and NS5 were determined by western blot in parallel with host cell GAPDH as a control. B – E Band intensities from A were quantitated and normalized to expression of GAPDH. All experiments were undertaken independently in triplicate. Error bars show + S.E.M. *p ≤ 0.05, **p ≤ 0.01). Uncropped western blots for all replicates are provided in the associated supplemental materials

Article Snippet: Membranes were subsequently blocked with 5% skim milk in TBS/0.05% Tween 20 at room temperature and subsequently incubated with a pan-specific mouse anti-flavivirus E protein monoclonal antibody from hybridoma HB112 [ ] at a 1:500 dilution, or a rabbit anti-dengue type 2 NS1 antibody at a 1:2000 dilution (PA5-32207; Thermo Scientific, Waltham, MA), or a rabbit polyclonal anti-DENV 2 NS3 antibody at a 1:8000 dilution (GTX124252; GeneTex Inc., Irvine, CA), or a mouse anti-DENV 2 NS5 monoclonal antibody at a 1:5000 dilution (MA5-17295, Thermo Fisher Scientific, Waltham, MA), or a 1:5000 dilution of mouse anti-GAPDH monoclonal antibody (sc-32233; Santa Cruz Biotechnology Inc., Dallas, TX) overnight at 4 °C.

Techniques: Infection, Solvent, Control, Western Blot, Expressing

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Mosquito metabolomics reveal that dengue virus replication requires phospholipid reconfiguration via the remodeling cycle

doi: 10.1073/pnas.2015095117

Figure Lengend Snippet: Impact of de novo PL pathway on DENV cellular cycle. Aag2 cells were supplemented with Etn or control media (Ctrl) for 24 h before and throughout infection with DENV at a MOI of 1. (A) Numbers of attached and internalized (+)gRNA. (B) NS3 protein level at 3, 4, 5, and 6 hpi. Actin was used for normalization. (C and D) Numbers of cellular (−)gRNA (C) and (+)gRNA (D) per well at 1, 3, 6, 9, 12, 15, 18, 21, and 24 hpi. (E) PFU per milliliter in supernatant at 32 hpi. (F) Number of (+)gRNA per milliliter in supernatant at 32 hpi. (G) Ratio of (+)gRNA/PFU in supernatant at 32 hpi. (A, C, D, and F) Lines show geometric means ± 95% CI. Four (A) and six (B) biological repeats were collected from independent wells. (B, E, and G) Lines show mean ± SEM (C and D) Tukey box plots. (C, D, and E–G) Six biological replicates were collected from two independent experiments. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001, as determined by Dunnett’s multiple comparisons test (A–D), an unpaired t test (E and F), or the Mann–Whitney U test (G).

Article Snippet: Antibodies used were anti-DENV-2 NS3 (CTX124252, Genetex) and anti-beta-actin (SC-47778, Santa-Cruz Biotechnology). . Replication Assay.

Techniques: Control, Infection, MANN-WHITNEY

Journal: Journal of Virology

Article Title: TRIM69 Inhibits Vesicular Stomatitis Indiana Virus

doi: 10.1128/JVI.00951-19

Figure Lengend Snippet: TRIM69-mediated inhibition of DENV-2 was not detected under conditions that restrict VSIV. Vero cells were modified to stably express human, rat, and mouse TRIM69 or were transduced with the corresponding empty vector (SCRPSY). Where indicated, cells transduced with the empty vector were also pretreated with 1,000 units of IFN-β. Cells were then challenged with DENV-2 propagated in Vero cells (mammalian DENV-2) (A and B) or with DENV-2 produced from Aedes albopictus C6/36 cells (insect DENV-2) (C and D). At 48 h after infection, cells were fixed and stained for DENV-2 infection, and infected foci were imaged (representative images are shown panels B and D) and enumerated (A and C) using a Celigo imaging cytometer. (E and F) As described in the legend for panels A to D, Vero cells were infected with FL-VSIV-GFP, fixed, and analyzed 16 h after infection. (G) HEK 293T cells were modified to stably express human TRIM69 or were transduced with the corresponding empty vector (SCRPSY). Empty (−) and TRIM69-expressing (+) cells were transfected with DENV-2 NS3, DENV-2 NS3 K104R, or empty expression plasmids, and NS3 expression was analyzed 48 h later using Western blotting. (H) In parallel with the experiment shown in panel G, functional TRIM69 expression in equivalent HEK 293T cells was assessed by titrated infection with VSIV-GFP. In all cases, virus titrations were carried out on at least two occasions, and typical results are shown. Means and standard deviations are plotted.

Article Snippet: Blots were probed with either anti-actin (JLA20 hybridoma; courtesy of the Developmental Studies Hybridoma Bank, University of Iowa), one of two anti-TRIM69 antibodies (catalog no. ab111943 [Abcam] or PA5-12215 [Thermo Fisher]), anti-DENV-2 NS3 (PA5-32199; Thermo Fisher), anti-phospho-STAT1 (Tyr701) (58D6), or anti-c-myc (9E10 hybridoma; Developmental Studies Hybridoma Bank, University of Iowa).

Techniques: Inhibition, Modification, Stable Transfection, Transduction, Plasmid Preparation, Produced, Infection, Staining, Imaging, Cytometry, Expressing, Transfection, Western Blot, Functional Assay, Virus

Journal: Journal of Virology

Article Title: RPLP1 and RPLP2 Are Essential Flavivirus Host Factors That Promote Early Viral Protein Accumulation

doi: 10.1128/JVI.01706-16

Figure Lengend Snippet: RPLP1/2 are required for early viral protein accumulation. (A) Knockdown of RPLP1/2 is shown by Western blotting of A549 cells with or without NITD008 treatment. (B) Quantification of DENV-2 RNA by RT-qPCR normalized to 18S rRNA is shown under control and RPLP1/2 knockdown conditions. (C) Luciferase measurements from infected cells harvested 4 hpi are shown for each siRNA transfection condition in the presence or absence or NITD008 treatment. (D) A549 cells were transfected with NSC siRNA or siRNA against RPLP2 and then infected with DENV-2 for 6 h at an MOI of 10 in the presence or absence of NITD008. NS3 accumulation was detected by Western blotting, and results for triplicate samples are shown for NSC and siP2 conditions. CHX-treated and uninfected samples served as controls for background signal. (E) Quantification of NS3 levels was calculated by normalizing results to those for β-actin. The NSC condition result was set to 100%. Error bars represent standard deviation measurements of three independent wells. Statistical significance was assessed by a two-tailed Student's t test between NSC and experimental siRNA conditions. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001.

Article Snippet: Antibodies used were anti-RPLP1 ( Ab121190 ; Abcam), anti-RPLP2 ( Ab154958 ; Abcam), anti-mouse beta-actin (sc-47778; Santa Cruz Biotechnology), anti-DENV-2 NS3 (GTX124252; GeneTex), anti-DENV-2 E (GTX127277; GeneTex), and anti-DENV-2 C (GTX103343; GeneTex).

Techniques: Knockdown, Western Blot, Quantitative RT-PCR, Control, Luciferase, Infection, Transfection, Standard Deviation, Two Tailed Test