rabbit polyclonal antibody against tlr2  (Bioss)

Journal: PLoS ONE

Article Title: Stiffening-Induced High Pulsatility Flow Activates Endothelial Inflammation via a TLR2/NF-κB Pathway

doi: 10.1371/journal.pone.0102195

Figure Lengend Snippet: (A-B) High pulsatility flow (HPF), due to the use of a “stiff” tube upstream to cell culture, upregulated proinflammatory molecule mRNA (ICAM-1, VCAM-1, MCP-1 and E-selectin) and protein (MCP-1) in healthy PAECs, compared to low pulsatility flow (LPF) or to static conditions. (C-D) The mRNA and protein expression of TLR2 but not TLR4 in PAECs was highly upregulated by HPF. *: p<0.05 versus Static, †: p<0.05 versus LPF.

Article Snippet: Antibodies used here include: rabbit polyclonal antibody against bovine MCP-1 (1∶500 dilution; Kingfisher Biotech, St. Paul, MN), rabbit polyclonal antibody against TLR2 (1∶100 dilution; Bioss Inc, Woburn MA), mouse monoclonal antibody against TLR4 (1∶100 dilution; Acris Antibodies Inc, San Diego, CA) and mouse monoclonal antibody against GAPDH (1∶20,000 dilution; Sigma Inc, Saint Louis, MO).

Techniques: Cell Culture, Expressing

Journal: PLoS ONE

Article Title: Stiffening-Induced High Pulsatility Flow Activates Endothelial Inflammation via a TLR2/NF-κB Pathway

doi: 10.1371/journal.pone.0102195

Figure Lengend Snippet: (A, C) At the mRNA level, TLR2/4 inhibitor OxPAPC or TLR2 siRNA but not TLR4 inhibitor CLI-095, decreased PAEC expression of ICAM-1, VCAM-1, MCP-1 and E-selectin mRNAs under HPF; TLR4 siRNA decreased ICAM-1 and E-selectin but not VCAM-1 and MCP-1 mRNAs. “*”: p<0.05 versus LPF, “†”: p<0.05 versus HPF. (B, D) At the protein level, the MCP-1 expression in PAECs exposed to HPF was inhibited by OxPAPC or TLR2 siRNA treatment but not CLI-095 or TLR4 siRNA. The black line in the blot images (D, right) shows separated lanes obtained on the same gel.

Article Snippet: Antibodies used here include: rabbit polyclonal antibody against bovine MCP-1 (1∶500 dilution; Kingfisher Biotech, St. Paul, MN), rabbit polyclonal antibody against TLR2 (1∶100 dilution; Bioss Inc, Woburn MA), mouse monoclonal antibody against TLR4 (1∶100 dilution; Acris Antibodies Inc, San Diego, CA) and mouse monoclonal antibody against GAPDH (1∶20,000 dilution; Sigma Inc, Saint Louis, MO).

Techniques: Expressing

Journal: PLoS ONE

Article Title: Stiffening-Induced High Pulsatility Flow Activates Endothelial Inflammation via a TLR2/NF-κB Pathway

doi: 10.1371/journal.pone.0102195

Figure Lengend Snippet: Pulse flow modulates TLR2-induced NF-κB activation in BPAECs. (A) Representative fluorescent images and quantitative measures of NF-kBp65 staining (red) in PAECs show HPF stimulation of PAECs led to increased intranuclear translocation or activation of NF-κB, which was reduced by TLR2/4 inhibitor OxPAPC and TLR2 siRNA. Blue stains show the nuclei. The scale bar shows 50 µm. (B) HPF stimulation of PAECs increased the mRNA levels of IKKα and IKKβ, both of which were attenuated in siRNA-transfected cells with knockdown of TLR2. (C) NF-κB inhibitor (BAY 11–7082) decreased the MCP-1 expression by PAECs exposed to HPF. *: p<0.05 versus LPF, †: p<0.05 versus HPF.

Article Snippet: Antibodies used here include: rabbit polyclonal antibody against bovine MCP-1 (1∶500 dilution; Kingfisher Biotech, St. Paul, MN), rabbit polyclonal antibody against TLR2 (1∶100 dilution; Bioss Inc, Woburn MA), mouse monoclonal antibody against TLR4 (1∶100 dilution; Acris Antibodies Inc, San Diego, CA) and mouse monoclonal antibody against GAPDH (1∶20,000 dilution; Sigma Inc, Saint Louis, MO).

Techniques: Activation Assay, Staining, Translocation Assay, Transfection, Expressing

Journal: PLoS ONE

Article Title: Stiffening-Induced High Pulsatility Flow Activates Endothelial Inflammation via a TLR2/NF-κB Pathway

doi: 10.1371/journal.pone.0102195

Figure Lengend Snippet: (A) PAECs from calves with hypoxia-induced pulmonary hypertension (PH) show elevated expression of TLR2 and TLR4, compared to control (CO). *:p<0.05. (B) Both immunostaining and western blotting results show elevated MCP-1 expression by PH-ECs compared to CO-ECs from calves. “PA” indicates the lumen of a pulmonary artery. *:p<0.05. (C) Enhanced TLR2 expression in the pulmonary arterial endothelium of human with pulmonary arterial hypertension (PAH). Cryosections of human intra-lobar pulmonary arteries were immunostained with TLR2 (red fluorescence) and counterstained with DAPI (cell nuclei, blue). Elastic lamellae showed green auto-fluorescence.

Article Snippet: Antibodies used here include: rabbit polyclonal antibody against bovine MCP-1 (1∶500 dilution; Kingfisher Biotech, St. Paul, MN), rabbit polyclonal antibody against TLR2 (1∶100 dilution; Bioss Inc, Woburn MA), mouse monoclonal antibody against TLR4 (1∶100 dilution; Acris Antibodies Inc, San Diego, CA) and mouse monoclonal antibody against GAPDH (1∶20,000 dilution; Sigma Inc, Saint Louis, MO).

Techniques: Expressing, Immunostaining, Western Blot, Fluorescence

Journal: Science signaling

Article Title: MEG3-4 is a miRNA decoy that regulates IL-1β abundance to initiate and then limit inflammation to prevent sepsis during lung infection

doi: 10.1126/scisignal.aao2387

Figure Lengend Snippet: (A) Primary alveolar macrophages (AMs) and AECII were infected with PAO1 at a multiplicity of infection (MOI) of 20:1 for 1 hour, polymyxin B (100 μg/ml) was added, and cells were cultured for another 1 hour to kill bacteria outside of the cell membrane. Samples were collected at multiple time points over 48 hours, and the expression of MEG3–4 in AMs and AECII cells is time-dependent, as detected by qRT-PCR. (B) Nuclear and cytosolic expression of MEG3–4 in primary AMs and AECII cells was detected by qRT-PCR. lncRNA Xist and H19 were used as nuclear and cytoplasmic controls, respectively. (C) TLR2 and TLR4 expression in AMs from wild-type (WT), Tlr2−/−, and Tlr4−/− mice was measured by immunoblotting. (D) WT, Tlr2−/−, and Tlr4−/− mice (n = 3) were infected with 5 × 106 CFU of PAO1 per mouse for 24 hours. AMs were collected to assess MEG3–4 expression. (E and F) MH-S cells were pretreated with indicated signaling pathway activators (a) or inhibitors (i) for 4 hours and then infected for 2 hours with PAO1 at an MOI of 20:1. MEG3–4 expression before and after infection was analyzed by qRT-PCR. (G and H) MH-S cells were transfected with control siRNA [scrambled siRNA (siNC)] and NF-κB p65 siRNA (si-p65) for 48 hours, respectively, and then infected with PAO1 at 20:1 MOI for 2 hours. Expression and phosphorylation of NF-κB p65 were measured by immunoblotting, and MEG3–4 transcripts were detected by qRT-PCR. Data in (C) and (G) are representative of three independent mice or cell samples. Data in (A), (B), (D) to (F), and (H) are means ± SD for three independent mice or cell samples (Kruskal-Wallis test; *P ≤ 0.05 and **P ≤ 0.01). NS, no significant change.

Article Snippet: Mouse monoclonal antibodies against β-actin (sc-47778), TLR4 (sc-293072), p-ERK (sc-7383), ERK1/2 (sc-514302), p-JNK (sc-6254), JNK (sc-7345), p-NF-κB p50 (sc-33022), NF-κB p50 (sc-166588), NF-κB p65 (sc-8008), p-Stat3 (sc-8059), pro-caspase I (sc-56036), MAPK p38 (sc-7972), and p53 (sc-377567); goat polyclonal antibodies against cleaved IL-1β (sc-23460), IL-6 (sc-1265), TNF-α (sc-1349), and NLRC4 (sc-49395); rabbit polyclonal antibodies against TLR2 (sc-10739), Stat3 (sc-482), p-NF-κB p65 (sc-33020), pro-IL-1β (sc-7884), cleaved caspase I (sc-514), ASC (sc-22514), and Akt1 (sc-8312); and chicken polyclonal antibodies against IL-18 (sc-7954-Y) were obtained from Santa Cruz Biotechnology.

Techniques: Infection, Cell Culture, Expressing, Quantitative RT-PCR, Western Blot, Transfection

Journal: Cell Research

Article Title: Type I CRISPR-Cas targets endogenous genes and regulates virulence to evade mammalian host immunity

doi: 10.1038/cr.2016.135

Figure Lengend Snippet: PA14 CRISPR-Cas regulates innate immunity through TLR4/NF-κB signaling. (A) MH-S cells were infected with PA14 WT and ΔTCR mutant at an MOI of 20:1 for 2 h. qPCR primer assay of immune response-related factors in ΔTCR mutant-infected MH-S cells versus PA14 WT-infected controls. Red dots indicate increased genes and green dots indicate decreased genes (over 10-fold change and P ≤ 0.05, n = 2 biological replicates). (B) Expression of TLR2, TLR4 and indicated signaling factors in PA14 WT-, ΔTCR mutant- and complemented strain-infected MH-S cells (30 min). (C) MH-S cells were infected with PA14 WT, ΔTCR mutant and complemented strain at an MOI of 20:1 for 0, 15, 30 and 60 min. CSLM results show the translocation of p-NF-κB (p-p65) in MH-S cells using immune staining. DAPI was used for staining the nucleus (arrows showing the nuclear translocation). (D) CSLM results show the production of TLR4 in MH-S cells. (E) TLR4 expression was determined in C57BL/6J WT and TLR4 KO mice by immunoblotting. (F) Secretion of TNF-α, IL-1β and IL-6 in BALF from C57BL/6J WT and TLR4 KO mice after WT PA14, ΔTCR mutant and complemented strain infection. (G) Phagocytosis of indicated MH-S cells was measured by CFU count assay. MH-S cells were infected with an MOI of 20:1, 25:1, 30:1, 35:1 and 40:1 PA14 WT. The MOI of 20:1 ΔTCR mutant infection was a control. (H) Expression of TLR4 and production of TNF-α, IL-1β and IL-6 in PA14 WT-infected and ΔTCR mutant-infected MH-S cells were detected by immunoblotting. Data are representative of three experiments expressed as means SEM (n = 3, one-way ANOVA with Tukey's post hoc; *P ≤ 0.05; **P ≤ 0.005).

Article Snippet: Mouse monoclonal Abs against β-actin (sc-47778), TLR4 (sc-293072), NF-κB p50 (sc-166588), p-NF-κB p50 (sc-33022), Stat6 (sc-374021), and Jak2 (sc-390539), rabbit polyclonal Abs against TLR2 (sc-10739), and p-Jak2 (sc-21870), goat polyclonal Abs against TNF-α (sc-1349), IL-6 (sc-1265), IL-1β (sc-23460) and p-Stat6 (sc-11762) were obtained from Santa Cruz Biotechnology (Dallas, TX).

Techniques: CRISPR, Infection, Mutagenesis, Expressing, Translocation Assay, Staining, Western Blot, Control