Hrp linked anti mouse igg antibody

Western blotting and pull-down assay were performed as described previously [12 (link),15 (link)]. Anti-monomeric Kusabira-Green N-terminal fragment antibodies (M148-3M), anti-DDDDK-tag monoclonal antibodies (FLA-1), and anti-S tag polyclonal antibodies were purchased from Medical & Biological Laboratories Co., Ltd., and anti-actin antibodies (AC-15) was obtained from Santa-Cruz Biotechnology, Inc. (Dallas, TX, USA). HRP linked anti-mouse IgG antibody (GE healthcare UK Ltd., Buckinghamshire, UK or Jackson ImmunoResearch, Inc., West Grove, PA, USA) and HRP linked anti-rabbit IgG antibody (Jackson ImmunoResearch, Inc.) were used as the secondary antibody. Antibody-bound proteins were visualized with ECL Western Blotting Detection Reagents (GE healthcare UK Ltd. or ATTO, Tokyo, Japan) on X-ray film (Fujifilm-Wako, Tokyo, Japan). For pull-down assays, transfected 293T cells were lysed by brief sonication in the presence of lysis buffer [20 mM HEPES (pH 7.9), 0.18 M NaCl, 0.1% NP-40, 0.1 mM EDTA, 10% glycerol, and cOmplete, Mini, EDTA-free Protease Inhibitor Cocktail (Roche Diagnostics GmbH, Mannheim, Germany)]. The cell extracts were subjected to affinity purification using S-protein-agarose (Merck Millipore, Burlington, MA, USA). Purified proteins and whole-cell extracts were subjected to western blotting.

Human placental extracts was provided from the Korean Pharmacopuncture Institute (Seoul, Korea). Primary antibodies specific for bromodeoxyuridine (BrdU), proliferating cell nuclear antigen (PCNA), fibroblast growth factor-7 (FGF-7), ß-actin were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA). Biotinylated goat anti-mouse immunoglobulin G (IgG) and avidin-biotin peroxidase complex were purchased from Vector Laboratories, Inc. (Burlingame, CA, USA). HRP-linked anti-mouse Ig G antibody was purchased from GE Healthcare Life Science (Pittsburgh, PA, USA). 3-3'diaminobenzidine, PBS solution, and hydrogen peroxide were purchased from Sigma-Aldrich Co. (Youngin, Korea). Trizol reagent and a reverse transcriptase-polymerase chain reaction (RT-PCR) kit were purchased from Invitrogen (Carlslab, CA, USA) and Bioneer Co. (Daejeon, Korea), respectively.