Journal: Scientific Reports

Article Title: Monocytes from patients with Primary Ciliary Dyskinesia show enhanced inflammatory properties and produce higher levels of pro-inflammatory cytokines

doi: 10.1038/s41598-017-15027-y

Figure Lengend Snippet: Chemoattractant receptor expression levels on monocytes of PCD patients and healthy controls. Expression levels of ( a ) CCR1, ( b ) CCR2, ( c ) CCR5, ( d ) FPR1, ( e ) BLT1 and ( f ) C5aR on monocytes of PCD patients (PCD), age-matched healthy pediatric controls (Ped CO) and healthy adult controls (Ad CO) were measured by flow cytometry. The chemoattractant receptor expression levels (mean fluorescence intensity, MFI) were normalized to the levels on monocytes of the reference Ad CO (%). Each dot represents the normalized MFI of one patient or healthy control (*p < 0.05, **p < 0.01; Mann Whitney U-test).

Article Snippet: Anti-CD14 [PE mouse anti-human, clone M5E2], anti-CD16 [PE mouse anti-human, clone 3G8], anti-CCR1 [Alexa Fluor 647 mouse anti-human CD191, clone 53504], anti-CCR2 [Alexa Fluor 647 mouse anti-human CD192, clone 48607], κ Isotype Control Alexa Fluor 647 [mouse IgG2b, clone 27–35], anti-BLT1 [mouse anti-human, clone 14F11], anti-FPR1 [mouse anti-human, clone 556015], anti-C5aR [mouse anti-human, clone D53–1472] and polyclonal PE goat anti-mouse were purchased from BD Biosciences (Temse, Belgium).

Techniques: Expressing, Flow Cytometry, Fluorescence, MANN-WHITNEY

Journal: Lipids in Health and Disease

Article Title: Critical role of leukotriene B 4 receptor signaling in mouse 3T3-L1 preadipocyte differentiation

doi: 10.1186/1476-511X-12-122

Figure Lengend Snippet: Expression of BLT1 and BLT2 during preadipocyte differentiation. Detection of BLT1 and BLT2 was performed by western blot analysis using anti-BLT1 or -BLT2 antibodies. GAPDH was used as an internal standard for confirmation of equally applied amounts of protein.

Article Snippet: Anti-BLT1 and -BLT2 polyclonal antibodies were purchased from Cayman Chemicals (Ann Arbor, MI, USA).

Techniques: Expressing, Western Blot

Journal: Lipids in Health and Disease

Article Title: Critical role of leukotriene B 4 receptor signaling in mouse 3T3-L1 preadipocyte differentiation

doi: 10.1186/1476-511X-12-122

Figure Lengend Snippet: Effect of BLT1 and BLT2 knockdown by siRNA on mouse 3T3-L1 preadipocyte differentiation. (A) : Confirmation of BLT1 and BLT2 knockdown by specific siRNAs. Negative control is cells treated with negative control siRNA (Stealth RNAi Negative Control Duplexes). (B) and (C) : Effect of BLT1 knockdown by siRNA. Representative microscopic (B) images of differentiated mouse 3T3-L1 adipocytes by Oil Red O staining. Scale bar represents 100 μm. (C) : Accumulation of TG in mature adipocytes was measured and expressed as TG contents (μg/mg protein). Each column represents the mean ± SEM from 3-5 independent experiments. * P <0.05 vs. negative control-siRNA treatment. (D) and (E) : Effect of BLT2 knockdown by siRNA. Representative microscopic (D) images of differentiated mouse 3T3-L1 adipocytes by Oil Red O staining. Scale bar represents 100 μm. (E) : Accumulation of TG in mature adipocytes was measured and expressed as TG contents (μg/mg protein).

Article Snippet: Anti-BLT1 and -BLT2 polyclonal antibodies were purchased from Cayman Chemicals (Ann Arbor, MI, USA).

Techniques: Negative Control, Staining

Journal: Lipids in Health and Disease

Article Title: Critical role of leukotriene B 4 receptor signaling in mouse 3T3-L1 preadipocyte differentiation

doi: 10.1186/1476-511X-12-122

Figure Lengend Snippet: Combination knockdown of BLT1 and BLT2 by siRNA on mouse 3T3-L1 preadipocyte differentiation. Mouse 3T3-L1 preadipocytes were treated with a combination of BLT1-siRNA (12.5 nM) and BLT2-siRNA (12.5 nM). (A) : Representative microscopic images of differentiated mouse 3T3-L1 adipocytes by Oil Red O staining. Scale bar represents 100 μm. (B) : Accumulation of TG in mature adipocytes was measured and expressed as TG contents (% of negative control). Each column represents the mean ± SEM from 3 independent experiments. * P <0.05, ** P <0.01 vs. negative control-siRNA treatment.

Article Snippet: Anti-BLT1 and -BLT2 polyclonal antibodies were purchased from Cayman Chemicals (Ann Arbor, MI, USA).

Techniques: Staining, Negative Control

Journal: The Journal of Experimental Medicine

Article Title: Cysteinyl leukotrienes mediate lymphokine killer activity induced by NKG2D and IL-15 in cytotoxic T cells during celiac disease

doi: 10.1084/jem.20150303

Figure Lengend Snippet: CystLT synthesis and signaling in an autocrine manner plays a role in NKG2D-mediated cytotoxicity in IE-CTLs. (A) IE-CTLs were pretreated for 30 min with pharmacological antagonists of CystLTR1 or BLT1 at 10 µM (MK571 and U75, respectively) or vehicle control (DMSO) before the cytolysis assay performed against 51 Cr-labeled EL4-MICA target cells at an effector/target ratio of 18:1. Data are means ± standard deviation of three independent experiments using three different CTL lines. (B) TALL-104 cells were transfected with siRNA against CystLTR1 or BLT1 or with a scrambled control siRNA by electroporation. Efficacy of knockdown is shown by a representative Western blot at the top. Cells were allowed to recover for 24 h before incubation with 51 Cr-labeled targets at an effector/target ratio of 3:1. Data are means ± standard deviation of five independent experiments. (C) Human IE-CTLs were pretreated for 30 min with 10 µM MK886 or vehicle control, and rescue experiments were performed by adding 1 µM LTD4 or LTB4 after 1 h of co-culture of effector cells with EL4-MICA targets at an effector/target ratio of 18:1. The cytolytic assay was performed after an additional 3 h of incubation. Data are means ± standard deviation of three independent experiments using two different cell lines. (D and E) Schematic diagram of experimental design. Human IE-CTLs were stimulated for 4 h with plate-bound antibodies against NKG2D or an IgG control. Supernatants were collected and used to stimulate TALL-104 cells transfected with siRNA against LTC4S or CystLTR1 or a scrambled control siRNA in the presence of 51 Cr-labeled EL4-MICA targets at the indicated effector/target ratios. Two different human IE-CTL lines were used. Data are representative of three independent experiments. (F) Experimental design is as in E, but here the effector/target ratio is 12.5:1 only. Data are normalized to the cytotoxic capacity measured in TALL cells that were transfected with control siRNA and are presented as means ± standard deviation of three independent experiments. *, P < 0.05; **, P < 0.01; ***, P < 0.001.

Article Snippet: LTB4, LTD4, BLT1 antagonist U75302, polyclonal antibodies specific for 5-LO, polyclonal antibodies specific for CystLTR1, polyclonal antibodies specific for BLT1, and montelukast (sodium salt) were obtained from Cayman Chemical.

Techniques: Control, Labeling, Standard Deviation, Transfection, Electroporation, Knockdown, Western Blot, Incubation, Co-Culture Assay

Journal: Parasites, Hosts and Diseases

Article Title: Dynamin 2-mediated endocytosis of BLT1 is required for IL-8 production in HMC-1 cells induced by Trichomonas vaginalis -derived secretory products

doi: 10.3347/PHD.24049

Figure Lengend Snippet: BLTs is required for IL-8 secretion in HMC-1 cells induced by TvSP. (A) BLT1 protein expression levels by immunoblotting following silencing of BLT1 gene by siRNA in HMC-1 cells. (B) BLT2 protein levels by immunoblotting following silencing of BLT2 gene by siRNA in HMC-1 cells. At 72 h post-transfection, whole-cell lysate from HMC-1 cells transfected with vehicle alone (Mock), control scrambled siRNA (100 nM) or BLT1 siRNA (100 nM) were subjected to immunoblotting with anti-BLT1, anti-BLT2, or anti-β-actin antibody as loading control. Blots are representative of 3 independent experiments. (C) Effect of BLT1 siRNA transfection on TvSP-induced IL-8 secretion in HMC-1 cells. (D) Effect of BLT2 siRNA on TvSP-induced IL-8 secretion in HMC-1 cells. HMC-1 cells were stimulated for 16 h with or without TvSP. The amount of IL-8 in culture supernatant was measured by human IL-8 ELISA. Data are expressed as the mean±SD from 4 independent experiments. Significant differences from the value obtained with cells incubated with medium alone are shown. LTB 4 , leukotriene B 4 ; HMC-1, human mast cell line; TvSP, Trichomonas vaginalis -derived secretory products; siRNA, short interfering RNA; IL-8, interleukin-8; BLT1, high affinity LTB 4 receptor; BLT2, low affinity LTB 4 receptor. * P <0.05.

Article Snippet: Rabbit anti-BLT1 antibody (NBP2-27422) was purchased from Novus Biologicals (Centennial, CO, USA).

Techniques: Expressing, Western Blot, Transfection, Control, Enzyme-linked Immunosorbent Assay, Incubation, Derivative Assay, Small Interfering RNA

Journal: Parasites, Hosts and Diseases

Article Title: Dynamin 2-mediated endocytosis of BLT1 is required for IL-8 production in HMC-1 cells induced by Trichomonas vaginalis -derived secretory products

doi: 10.3347/PHD.24049

Figure Lengend Snippet: Kinetics of trafficking of BLT1 in HMC-1 cells stimulated for up to 60 min with TvSP (A) or LTB 4 (B). Cells were stained with anti-BLT1 antibody and then measured by Flow cytometry. LTB 4 was used as a positive control. Data are presented as the means±SD from 3 independent experiments. LTB 4 , leukotriene B 4 ; HMC-1, human mast cell line; TvSP, Trichomonas vaginalis -derived secretory products; BLT1, LTB 4 receptor. * P <0.05; ** P <0.01.

Article Snippet: Rabbit anti-BLT1 antibody (NBP2-27422) was purchased from Novus Biologicals (Centennial, CO, USA).

Techniques: Staining, Flow Cytometry, Positive Control, Derivative Assay

Journal: Parasites, Hosts and Diseases

Article Title: Dynamin 2-mediated endocytosis of BLT1 is required for IL-8 production in HMC-1 cells induced by Trichomonas vaginalis -derived secretory products

doi: 10.3347/PHD.24049

Figure Lengend Snippet: Effect of dynamin-2 siRNA transfection on internalization of BLT1 in HMC-1 cells induced by TvSP or LTB 4 . (A) Dynamin-2 protein levels by immunoblotting following silencing of dynamin-2 gene by siRNA in HMC-1 cells. At 72 h post-transfection, whole-cell lysate from HMC-1 cells transfected with vehicle alone (Mock), control scrambled siRNA (100 nM) or dynamin 2 siRNA (100 nM) were subjected to immunoblotting with anti-BLT1 or anti-β-actin antibody as loading control. Blots are representative of 3 independent experiments. Surface and intracellular expression of BLT1 in HMC-1 cells stimulated for up to 60 min with TvSP (B) or LTB 4 (C) cells were stained with anti-BLT1 antibody and then measured by Flow cytometry. LTB 4 was used as a positive control. Data are presented as the means±SD from 3 independent experiments. LTB 4 , Leukotriene B 4 ; HMC-1, Human mast cell line; TvSP, Trichomonas vaginalis -derived secretory products; siRNA, short interfering RNA; DNM2, dynamin-2; BLT1, LTB 4 receptor. ** P <0.01.

Article Snippet: Rabbit anti-BLT1 antibody (NBP2-27422) was purchased from Novus Biologicals (Centennial, CO, USA).

Techniques: Transfection, Western Blot, Control, Expressing, Staining, Flow Cytometry, Positive Control, Derivative Assay, Small Interfering RNA

Journal: Parasites, Hosts and Diseases

Article Title: Dynamin 2-mediated endocytosis of BLT1 is required for IL-8 production in HMC-1 cells induced by Trichomonas vaginalis -derived secretory products

doi: 10.3347/PHD.24049

Figure Lengend Snippet: Interaction of dynamin-2 with BLT1 in TvSP (A) or LTB 4 (B)-stimulated HMC-1 cells. HMC-1 cells (1×10 7 /sample) were stimulated with TvSP or LTB 4 for 30 or 60 min. Cells were precipitated with anti-dynamin-2 or BLT1 antibodies for 18 h and then blotted with anti-BLT1 or dynamin-2 antibodies. Interaction of dynamin-2 and BLT1 was evaluated by coimmunoprecipitation. Anti-β-actin antibody was used as loading control. The images are representative of 3 independent experiments with similar results. LTB 4 , Leukotriene B 4 ; HMC-1, human mast cell line; TvSP, Trichomonas vaginalis -derived secretory products; BLT1, LTB 4 receptor; DNM2, dynamin-2; IB, westernblotting after IP; WB, westernblotting in cell lysate.

Article Snippet: Rabbit anti-BLT1 antibody (NBP2-27422) was purchased from Novus Biologicals (Centennial, CO, USA).

Techniques: Control, Derivative Assay