mouse anti tlr2 ab  (Hycult Biotech)

Journal: Scientific Reports

Article Title: Recombinant Mtb9.8 of Mycobacterium bovis stimulates TNF-α and IL-1β secretion by RAW264.7 macrophages through activation of NF-κB pathway via TLR2

doi: 10.1038/s41598-018-20433-x

Figure Lengend Snippet: TLR2 is required for the secretion of TNF-α and IL-1β by rMtb9.8-induced mouse macrophages. RAW264.7 cells were pre-treated for 30 min at 37 °C with anti-TLR2 (30 µg/ml), anti-TLR4 (30 µg/ml) or isotype Ab (30 µg/ml) before stimulation with rMtb9.8 (5 μg/ml) for 30 min. LPS and Pam3CSK4 were used as positive controls to demonstrate the blocking effects of TLR4 and TLR2 antibodies. The levels of TNF-α ( A ) and IL-1β ( B ) in the supernatant were measured by ELISA. The data are expressed as the mean ± SD of the results of three separate experiments. **P < 0.01, ***P < 0.001, stimulated cells versus cells cultured in medium alone.

Article Snippet: In experiments designed to evaluate NF-κB activation by TLR2 or TLR4, RAW264.7 cells were pre-treated for 30 min at 37 °C with 30 μg/ml of a mouse Ab against TLR2, TLR4 or a mouse IgG isotype-matched control Ab (isotype Ab) (all from Imgenex). pNF-κB-Luc expression after rMtb9.8 stimulation was measured using a luciferase assay after transfection with the reporter plasmid, pNF-κB-Luc (0.2 μg/well).

Techniques: Blocking Assay, Enzyme-linked Immunosorbent Assay, Cell Culture

Journal: Scientific Reports

Article Title: Recombinant Mtb9.8 of Mycobacterium bovis stimulates TNF-α and IL-1β secretion by RAW264.7 macrophages through activation of NF-κB pathway via TLR2

doi: 10.1038/s41598-018-20433-x

Figure Lengend Snippet: TLR2 is required for NF-κB activation by rMtb9.8. ( A ) RAW264.7 cells were transfected with the dual luciferase assay reporter vector pNF-κB-luc and the Renilla luciferase reporter vector pRL-TK. Exposure of the cells to rMtb9.8 significantly induced luciferase activity (P < 0.01) in a time-dependent manner, and this increase was significantly attenuated (P < 0.01) in cells transfected with the dominant-negative mutant of the NF-κB p65 plasmid pp65RHD. RAW264.7 cells were pre-treated with a mouse Ab against TLR2 or TLR4 or with an isotype Ab, and the luciferase activity due to pNF-κB-Luc expression was measured after rMtb9.8 stimulation. The Ab against TLR2 significantly decreased the activation of NF-κB luciferase activity after rMtb9.8 treatment, but the antibody against TLR4 and the isotype Ab did not. The data shown are representative of at least three independent experiments and are reported as the mean ± SD (n = 3). Different letters (a,b) indicate significant differences between groups (P < 0.01). ( B ) HEK293T cells, HEK-TLR2 cells and HEK-TLR4 cells were transfected, and the dual luciferase assay was performed. Luciferase activity was significantly induced by rMtb9.8 in HEK-TLR2 cells compared with HEK-TLR4 cells or untreated cells (P < 0.001). The data shown are representative of at least three independent experiments and are reported as the mean ± SD (n = 3).

Article Snippet: In experiments designed to evaluate NF-κB activation by TLR2 or TLR4, RAW264.7 cells were pre-treated for 30 min at 37 °C with 30 μg/ml of a mouse Ab against TLR2, TLR4 or a mouse IgG isotype-matched control Ab (isotype Ab) (all from Imgenex). pNF-κB-Luc expression after rMtb9.8 stimulation was measured using a luciferase assay after transfection with the reporter plasmid, pNF-κB-Luc (0.2 μg/well).

Techniques: Activation Assay, Transfection, Luciferase, Plasmid Preparation, Activity Assay, Dominant Negative Mutation, Expressing