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For Western blot analysis, cells were lysed on ice in lysis buffer containing 25 mM Tris–HCl (pH 7.4), 150 mM NaCl, 1 mM EDTA, 5% glycerol, 0.5% Tween-20, 1 mM Na₃VO₄, 10 mM NaF, 1 mM PMSF, 1 mM benzamidine, 1 μg/ml pepstatin A, 5 μg/ml leupeptin, and 5 μg/ml aprotinin. Cell lysates were clarified by centrifugation at 20,000g at 4 °C for 15 min. Protein concentration was determined using Pierce BCA kit. Protein samples were boiled in 1× sample buffer and separated using SDS-PAGE and transferred to nitrocellulose membranes by semi-dry transfer for 1 h. Membranes were incubated at room temperature in 1% Casein in TBS (BioRad cat #1610782) blocking buffer followed by overnight incubation with primary antibodies (1:1000 in 5% BSA in TBST). Proteins were visualized after incubation with fluorescence secondary antibodies and image processing using LI-COR image Studio Software. Primary antibodies used from Cell Signaling were p38 MAPK (Thr180/Tyr182) (3D7) (cat# 9215), pERK1/2 (cat#9101), ERK1/2 (cat#9107), pSAPK/JNK (cat#4668), JNK1 (cat#3708), GFP (cat#2955) and from Sigma flag M2 (cat#F3165) and M2 (cat#F7425), and Roche: HA 12CA5 (cat#11583816001).

ST2 cells (3 × 10⁵ cells/mL) were cultured in a Φ 6 cm plate and stimulated with E. coli-LPS (1 μg/mL), EA (3 μg/mL). Western blotting was performed as described previously by Kudo et al. [35 (link)]. In brief, cell pellets were resuspended in ice-cold lysis buffer. Proteins were separated by SDS-PAGE, electro-blotted onto nitrocellulose membrane, and were visualized by the ECL western blotting detection system (GE Healthcare, UK) (Amersham, Piscataway, NJ, USA). The following antibodies from Cell Signaling Technology were used as primary antibodies: p-NF-κB p65 (#3033; 1 : 1,000), NF-κB p65 (#8242; 1 : 1,000), p-p38mitogen-activated protein kinase (p-p38 MAPK) (#4511; 1 : 1,000), p38 MAPK (#8690; 1 : 1,000), p-SAPK/JNK (#4668; 1 : 1,000), t-SAPK/JNK (#9258; 1 : 1,000), p-IKKα/β (S176/180) (#2697S; 1 : 1,000), IKKβ (#2678; 1 : 1,000), and β-actin (#A2228; 1 : 8,000; Sigma-Aldrich). The following antibody from GeneTex was used as the primary antibody: beta catenin antibody [N1N2-2], N-term (#GTX101435; 1 : 1,000).