Goat anti rabbit igg

Lung tissues were homogenized in lysis buffer containing RIPA II cell lysis buffer (GenDEPOT, Barker, TX), protease inhibitors and phosphatase inhibitors and put on the ice for 20 min. These lysed proteins were centrifuged for 15 min. Thirty microgram of protein from supernatant was separated by SDS-PAGE and transferred to PVDF membrane (Bio-Rad Laboratories, Hercules, CA). After blocking with 5% skim milk (Bio-Rad), membrane was incubated with 2 µl primary antibodies against Bax, Bcl-2, Caspase-3, Caspase-9 (Cell Signaling Technology, Denver, MA for all), AT₁R (Sigma-Aldrich Co.), AT₂R (Sigma-Aldrich Co.), Mas receptor (Alomone Labs, Israel), ACE (Santa Cruz Biochemicals, Santa Cruz, CA), and GAPDH (Bioworld technology, St. Louis Park, MN). After washed with T-TBS buffer 6 times for 5 min each, membrane was incubated with goat anti-rabbit IgG (Enzo Life Sciences) for 30 min. Membrane was then washed again with T-TBS, developed using ECL (Amersham, Arlington, IL) and visualized using LAS-1000 (Fujifilm, Japan).

Rhizomes of A. macrocephala were purchased from Kimitongsang (Seoul, Korea) in April 2009 and authenticated by Prof. K. S. Yang at College of Pharmacy, Sookmyung Women’s University. A voucher specimen (No. SPH 09003) was deposited in the herbarium of Sookmyung Women’s University. β-catenin antibody were purchased from BD Transduction Laboratories (San Jose, CA, USA). Galectin-3 antibody was purchased from Santa Cruz Biotechnology (Dallas, Texas, USA). Lamin A/C and cyclin D1 antibodies were purchased from Cell Signaling (Danvers, MA, USA), and β-actin antibody was from Sigma-Aldrich (St Louis, MO, USA). HRP-conjugated goat anti-mouse IgG and goat anti-rabbit IgG was purchased from Enzo Life Science (East Farmingdale, NY, USA). Other chemical reagents including 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT), were purchased from Sigma-Aldrich.

Thiazolyl blue tetrazolium bromide (MTT), 2″,7″-dichlorodihydro-fluorescein diacetate (DCFH-DA) and other reagents were purchased from Sigma-Aldrich Co. (St. Louis, MO, USA). Tris was purchased from Duchefa Biochemie (BH Haarlem, The Netherlands), dimethyl sulfoxide (DMSO) from Junsei Chemical Co. (Tokyo, Japan). Fetal bovine serum (FBS) and penicillin-streptomycin (P/S) were purchased from Gibco (Los Angeles, CA, USA). Dulbecco's Modified Eagle's Medium (DMEM) was purchased from Welgene Inc. (Gyeongsangbuk-do, Korea). BCA reagent and albumin standard were from Thermo Scientific (Waltham, MA, USA). Primary Bcl-2 antibody was purchased from Oncogene (Bracknell, England). Bax and PAPR antibodies were obtained from Cell Signaling (Danvers, MA, USA) and cytochrome c, caspase-9, and β-actin antibodies from Santa Cruz (Paso Robles, CA, USA). Caspase-3 antibody was obtained from EMD Millipore (Billerica, MA, USA). Secondary antibodies goat anti-rabbit IgG, pAb, goat anti-mouse IgG, pAb, and caspase-3 activity assay kit were obtained from Enzo Life Sciences (Farmingdale, NY, USA). West-Q chemiluminescent substrate was purchased from GenDEPOT (Katy, TX, USA). The solvents used were of HPLC grade, unless stated otherwise.