Journal: Scientific Reports
Article Title: Resveratrol inhibits IL-33–mediated mast cell activation by targeting the MK2/3–PI3K/Akt axis
Figure Lengend Snippet: Inhibition of IL-33–induced mast cell activation via blockade of the MK2−PI3K−Akt pathway by resveratrol. ( A ) Western blot analysis of phospho–TAK1, phospho–p38, phospho–MK2, and phospho–Akt in BMMCs stimulated with 1 ng/ml IL-33 for up to 20 min in the presence or absence of 25 μM resveratrol or TAK1 inhibitor 5Z-7-Ox. The level of β-actin is shown at the bottom as a loading control. ( B ) In vitro p38 and MK2 kinase assay using p38 and MK2 positive controls (n = 3). SB: 10 μM SB203580, PF: 10 μM PF-3644022. ( C ) Western blot analysis of phospho–Akt in BMMCs stimulated with IL-33 for up to 20 min in the presence or absence of resveratrol, SB, or PF. The level of β-actin is shown at the bottom as a loading control. ( D ) ELISA of IL-6, IL-13, and TNF-α in BMMCs treated with IL-33 for 6 h in the presence or absence of resveratrol, 10 µM SB, or 10 μM PF (n = 3). ( E ) ELISA of IL-6, IL-13, and TNF-α in BMMCs treated with IL-33 for 6 h in the presence or absence of resveratrol, 5 μM LY294002 (LY), or 100 nM wortmannin (WM) (n = 3). * P < 0.05, *** P < 0.001, **** P < 0.0001; N.D., not detected.
Article Snippet: Reagents used in this study were acquired from the indicated suppliers: trans-resveratrol, cyclodextrin, 5Z-7-Oxozeaenol and Evans blue (Sigma-Aldrich, St. Louis, MO, USA); LY294002, wortmannin, and ICI 182,780 (Abcam, Cambridge, UK); PF-3644022 (TOCRIS Bioscience, Bristol, U.K.); recombinant mouse IL-3, recombinant mouse stem cell factor (SCF), and recombinant human IL-33 (PeproTech, Rocky Hill, NJ, USA); recombinant human IL-3 (Thermo Fisher Scientific, Wilmington, DE, USA); recombinant mouse IL-33 (R & D systems, Minneapolis, MN, USA); anti-TNP IgE, anti–DNP mouse IgE mAb, anti–mouse CD16/32, PE–conjugated anti–mouse c-kit Ab, and APC–conjugated anti–mouse ST2 Ab (BD Bioscience, San Jose, CA, USA); DNP–BSA (Cosmo Bio, Tokyo, Japan); APC–conjugated anti–mouse CD63 Ab (Miltenyi Biotec, Bergisch Gladbach, Germany); anti–phospho–transforming growth factor β-activated kinase 1 (TAK1) Ab (Thr184/187; #4508), anti–phospho–IκB kinase (IKK) α/β Ab (Ser176/177; #2078), anti–phospho–p65 Ab (Ser536; #3033), anti–phospho–p38 Ab (Thr180/Thy182; #4511), anti–phospho–MK2 Ab (Thr334; #3007), anti–phospho–Akt Ab (Ser473; #4060), anti–phospho–Gab2 Ab (Tyr452; #3882), anti–phospho–Syk Ab (Tyr525/526; #2710), anti–phospho–p70S6K Ab (Tyr389; #9234), anti–phospho–AMPK Ab (Thr172; #2535), anti–β-actin Ab (#4970), and AICAR (#9944) (Cell Signaling Technology, Danvers, MA, USA).
Techniques: Inhibition, Activation Assay, Western Blot, In Vitro, Kinase Assay, Enzyme-linked Immunosorbent Assay