Journal: Journal of Cellular and Molecular Medicine

Article Title: Interleukin-33 overexpression is associated with liver fibrosis in mice and humans

doi: 10.1111/j.1582-4934.2009.00801.x

Figure Lengend Snippet: Antibodies used

Article Snippet: FITC-conjugated mouse monoclonal IgG1 anti-human ST2L (clone B4E6) , 10 μq/ml , MD Biosciences (Zürich, Switzerland).

Techniques:

Journal: Investigative Ophthalmology & Visual Science

Article Title: IL-33 Shifts Macrophage Polarization, Promoting Resistance against Pseudomonas aeruginosa Keratitis

doi: 10.1167/iovs.09-3983

Figure Lengend Snippet: Primer Sequences for Real-time PCR

Article Snippet: After blocking with 0.01 M phosphate buffer containing 2.5% BSA and goat IgG (1:100) for 3 minutes at room temperature, sections were incubated with primary antibodies, rat anti-mouse Mφ (1:100; Santa Cruz Biotechnology, Santa Cruz, CA), rabbit anti-mouse NOS2 (1:100; Santa Cruz Biotechnology), rabbit anti-mouse Arg1 (1:100; Santa Cruz Biotechnology), or rabbit anti-mouse ST2 (2 μg/mL; ProSci Incorporated, Poway, CA) for 1 hour, followed by Alexa Fluor 546-conjugated goat anti-rat antibody (1:1500; Invitrogen) or Alexa Fluor 633-conjugated goat anti-rabbit antibody (1:1500; Invitrogen) for another hour.

Techniques:

Journal: Investigative Ophthalmology & Visual Science

Article Title: IL-33 Shifts Macrophage Polarization, Promoting Resistance against Pseudomonas aeruginosa Keratitis

doi: 10.1167/iovs.09-3983

Figure Lengend Snippet: Modulation of IL-33 in vitro. Real-time RT-PCR showed ST2 expression on RAW cells was significantly upregulated over constitutive levels after stimulation with LPS after 2 (P < 0.05), 4, 6, and 8 hours (each at P < 0.01) (A). After transfection with a vector overexpressing IL-33, RAW cells significantly downregulated mRNA for pro-inflammatory mediators (B) IL-1β (P < 0.05), MIP-2 (P < 0.01), IL-6 (P < 0.01), and TNF-α (P < 0.01). In addition, Th1-type cytokines such as IFN-γ (P < 0.01) and IL-12 (P < 0.01) (C) and Th2-type cytokines such as IL-5 (P = 0.01) and IL-10 (P < 0.01) (D) were upregulated. Data are the mean ± SEM of two similar experiments (5 samples/group/time).

Article Snippet: After blocking with 0.01 M phosphate buffer containing 2.5% BSA and goat IgG (1:100) for 3 minutes at room temperature, sections were incubated with primary antibodies, rat anti-mouse Mφ (1:100; Santa Cruz Biotechnology, Santa Cruz, CA), rabbit anti-mouse NOS2 (1:100; Santa Cruz Biotechnology), rabbit anti-mouse Arg1 (1:100; Santa Cruz Biotechnology), or rabbit anti-mouse ST2 (2 μg/mL; ProSci Incorporated, Poway, CA) for 1 hour, followed by Alexa Fluor 546-conjugated goat anti-rat antibody (1:1500; Invitrogen) or Alexa Fluor 633-conjugated goat anti-rabbit antibody (1:1500; Invitrogen) for another hour.

Techniques: In Vitro, Quantitative RT-PCR, Expressing, Transfection, Plasmid Preparation

Journal: Investigative Ophthalmology & Visual Science

Article Title: IL-33 Shifts Macrophage Polarization, Promoting Resistance against Pseudomonas aeruginosa Keratitis

doi: 10.1167/iovs.09-3983

Figure Lengend Snippet: Immunostaining and real-time RT-PCR. Dual immunostaining for F4/80 (Mφ) and ST2 in the cornea of rmIL-33 compared with PBS-treated B6 mice. More staining for ST2 (blue) was associated with Mφ (red) in rmIL-33–-treated mice (A) than in PBS-treated mice (B) cornea. Substitution of the primary antibodies with IgG showed no detectable staining (C). Original magnification, ×1000. Real-time RT-PCR analysis of LPS-stimulated peritoneal-elicited Mφ, with or without rmIL-33, showed a significant upregulation of ST2 on Mφ after LPS stimulation in the presence of rmIL-33 (P = 0.04) (D). Upregulation of IL-10 (E; P = 0.003) and downregulation of IL-12 (F; P = 0.003) were observed after LPS, together with rmIL-33 treatment. Data for RT-PCR are the mean ± SEM of two similar experiments (5 samples/group/time).

Article Snippet: After blocking with 0.01 M phosphate buffer containing 2.5% BSA and goat IgG (1:100) for 3 minutes at room temperature, sections were incubated with primary antibodies, rat anti-mouse Mφ (1:100; Santa Cruz Biotechnology, Santa Cruz, CA), rabbit anti-mouse NOS2 (1:100; Santa Cruz Biotechnology), rabbit anti-mouse Arg1 (1:100; Santa Cruz Biotechnology), or rabbit anti-mouse ST2 (2 μg/mL; ProSci Incorporated, Poway, CA) for 1 hour, followed by Alexa Fluor 546-conjugated goat anti-rat antibody (1:1500; Invitrogen) or Alexa Fluor 633-conjugated goat anti-rabbit antibody (1:1500; Invitrogen) for another hour.

Techniques: Immunostaining, Quantitative RT-PCR, Staining, Reverse Transcription Polymerase Chain Reaction

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Focal adhesion kinase-mediated activation of glycogen synthase kinase 3β regulates IL-33 receptor internalization and IL-33 signaling

doi: 10.4049/jimmunol.1401414

Figure Lengend Snippet: A. MLE12 cells were transfected with ST2L-Flag plasmid (2 µg) for 2 days and then treated with IL-33 (0, 5, 10, and 20 ng/ml) for 30 min. Cell lysates were subjected to immunoprecipitation with an antibody against IgG or phospho(p)-serine, followed by immunoblotting with a Flag antibody. Input lysates were analyzed by immunoblotting with Flag and β-actin antibodies. B. RAW264 cells were treated with IL-33 (10 ng/ml, 30 min). Cell lysates were subjected to immunoprecipitation with an antibody against GSK3β, followed by immunoblotting with a ST2L antibody. Input lysates were immunoblotted with ST2L and β-actin antibodies. C. MLE12 cells were transfected with ST2L-Flag plasmid (2 µg) for 2 days and then treated with IL-33 for 0, 5, 15, and 30 min. Cell lysates were subjected to immunoprecipitation with an antibody against IgG or Flag, followed by immunoblotting with a GSK3β antibody. Input lysates were analyzed by immunoblotting with GSK3β, Flag, and β-actin antibodies. D. MLE12 cells grown on glass-bottom dishes were treated with IL-33 (10 ng/ml, 30 min). Immunostaining was performed to examine the localization of ST2L (green) and phospho-Y216-GSK3β (p-GSK3β, red). Representative images from over 40 cells, with > 80% of these staining positive, in three independent experiments are shown.

Article Snippet: FITC conjugated ST2L (FITC-ST2L) antibody (DJ8) and a Rat FITC conjugated IgG1 isotope control antibody were from MD Bioproducts (St. Paul, MN).

Techniques: Transfection, Plasmid Preparation, Immunoprecipitation, Western Blot, Immunostaining, Staining