Goat anti vcam 1

HUVECs were stimulated with TNFα for 18h and cells were lysed in lysis buffer, pH 7.4 (in mM) 50 HEPES, 5 EDTA, 120 NaCl), 1% Triton X-100, protease inhibitors (10 μg/ml aprotinin, 1 mmol/L phenylmethylsulfonyl fluoride, 10 μg/ml leupeptin) and phosphatase inhibitors (mmol/L) 50 sodium fluoride, 1 sodium orthovanadate, 10 sodium pyrophosphate). Cell lysates were used for immunoblotting, as described previously30 (link). Goat anti-ICAM-1 (Santa Cruz), goat anti-VCAM-1 (Santa Cruz), and Mouse anti-actin (Santa Cruz) were incubated on nitrocellulose membranes containing protein.
For protein expression in ischemic muscle, mice were perfused with cold phosphate buffer saline. Muscle samples were harvested and frozen in liquid nitrogen. Muscle samples were crushed and lysed with RIPA lysis buffer (5 mM Tris-HCl (pH 7.6), 150 mM NaCl, 1% NP-40, 1%sodium deoxycholate, 0.1% SDS) with protease inhibitor followed by brief sonication as described previously40 (link). Equal amount of protein was separated by SDS-PAGE. Following primary antibodies were used: anti-Atox1 (home-made), anti-p47phox (Santa Cruz), or anti-VEGF antibody (Abcam). Protein expression was visualized by ECL (Amersham). Band density was quantified by ImageJ.

Ferulic acid and 5-hydroxymethyl-2-furaldehyde (5-HMF) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Albiflorin and paeoniflorin were the products of Wako (Osaka, Japan). Nodakenin was purchased from NPC BioTechnology Inc. (Daejeon, Korea). The purity of all reference standards was ≥98.0%. HPLC-grade methanol, acetonitrile, and water were obtained from J.T.Baker (Phillipsburg, NJ, USA). Glacial acetic acid, analytical reagent grade, was purchased from Junsei (Tokyo, Japan). RPMI 1640, fetal bovine serum, TNF-α, tissue culture reagents, 2′,7′-bis(2-carboxyethyl)-5(6)-carboxyfluorescein acetoxy-methylester (BCECF-AM), DAF-FM diacetate, and CM-H₂DCFDA, Alexa Fluor 488 and 594 conjugated second antibodies were purchased from Invitrogen (San Diego, CA). Biotin 3′ End DNA Labeling Kit, LightShift® Chemiluminescent EMSA Kit, Biodyne® Precut Nylon Membranes, Lipofectamine LTX reagent, and Renilla-Firefly Luciferase Dual Assay Kit were purchased from Pierce Biotechnology (Rockford, USA). Primary antibodies, including mouse anti-ICAM-1, goat anti-VCAM-1, rabbit anti-E-selectin, mouse anti-NF-κB, mouse anti-p-IκB-α, rabbit anti-HO-1, and rabbit anti-Nrf2, were purchased from Santa Cruz Biotechnology (CA, USA). Donkey anti-goat IgG-H+I were purchased from Bethyl (Montgomery, USA) and goat anti-rabbit IgG and goat anti-mouse IgG were purchased from Enzo (Farmingdale, USA).