Phos p38 mapk

Brush border membrane (BBM) from IEC-18 cells for Western blot analysis was prepared as described earlier [27 (link)]. BBM and total cell lysate of IEC-18 cells were solubilized in RIPA buffer (Santa Cruz, Dallas, TX, USA) containing Halt protease inhibitor cocktail (Pierce, Thermo Scientific, Waltham, MA, USA) and cellular protein concentration was measured at A280 nm. Equal amounts of protein samples (150 µg) were resolved using 10% SDS PAGE and immobilized to Immobilon-P membranes (Millipore, Burlington, MA, USA). These membranes were probed with SGLT1 (Abcam, Cambridge, MA, USA), Ezrin (Millipore, Burlington, MA, USA), p38 MAPK, and phos p38 MAPK (Cell Signaling) specific antibodies at 1:1000 dilutions. Secondary detection using goat anti-rabbit Alexa 680 (Invitrogen, Carlsbad, CA, USA) was performed at 1:10,000 dilutions. Membranes were scanned using Odyssey Infrared Imaging System (LICOR Bioscience, Lincoln, NE, USA).

Dulbecco's modified Eagle's medium (DMEM), fetal bovine serum (FBS), and penicillin/streptomycin were obtained from Gibco/BRL Life Technologies Inc. (Grand Island, NY, USA). Lipopolysaccharide (LPS), 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), and 2′,7′-dihydrofluorescein diacetate (DCFH₂-DA) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Antibodies against iNOS, COX-2, MMP-2, MMP-9, t-PA, VEGF, PDGF, VCAM-1, and β-actin were purchased from Santa Cruz (Heidelberg, Germany). Antibodies against I-κBα, NF-κB, phos-ERK1/2, ERK1/2, phos-JNK, JNK, phos-p38 MAPK, and p38 MAPK, were obtained from Cell Signaling Technology Inc. (Danvers, MA, USA). Anti-p47^phox antibody was purchased from Merk-Millipore (Darmstadt, Germany). All other chemicals were reagent or HPLC grade and supplied by either Merck (Darmstadt, Germany) or Sigma (St. Louis, MO, USA).

Antcin A, antcin B, antcin C, antcin H, antcin K and antcin M were isolated from the fruiting bodies of A. cinnamomea and A. salmonea as described previously [17 (link)]. The purity of the antcins was above 99% as confirmed by HPLC and FT-NMR analysis. Minimum essential medium (MEM), Medium 199 (M-199), fetal bovine serum (FBS), sodium pyruvate, penicillin and streptomycin were obtained from Invitrogen (Carlsbad, CA). Heparin sodium salt, endothelial cell growth supplement (ECGS), N-acetylcysteine, 2′, 7′-dichlorofluorescein diacetate (DCFH₂-DA), 3-(4,5-dimethyl-thiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) and D-Glucose were purchased from Sigma-Aldrich (St Louis, CA). Antibodies against cyclin D1, cyclin B1, cyclin E, CDK2, CDK4, CDK6, Cdc2, phos-pRb, p16^INK4A, p21^CIP1, acety-p52, phos-p53, phos-FoxO1, FoxO1, phos-JNK/SAPK, JNK/SAPK, phos-p38 MAPK, p38 MAPK, phos-ERK1/2, ERK1/2, Phos-AKT, AKT, histone H3, phos-SIRT-1, SIRT-1, SIRT-3, SIRT-6 and Keap-1 were obtained from Cell Signaling Technology, Danvers, MA. Antibodies against p53, SMP30 and acetyl-FoxO1 were purchased from Santa Cruz Biotechnology, Dallas, TX. Antibodies against HO-1 and NQO-1 were obtained from Abcam, Cambridge, UK. All other chemicals were reagent grade or HPLC grade and supplied by either Merck (Darmstadt, Germany) or Sigma-Aldrich.