Anti rabbit horseradish peroxidase conjugated antibody

The MC3T3-E1 preosteoblast cells were cultured in medium supplemented with BetA (10 μM) and/or BMP2 (200 ng/ml). After 5 to 10 min, cells were harvested in a lysis buffer (Cell Signaling, Beverly, MA, USA) and the protein concentration was determined by the BCA assay reagent (Bio-Rad Laboratories, Hercules, CA, USA). Proteins were resolved on 10 % SDS-PAGE and transferred to a PVDF membrane. After blocking in 5 % skim milk in Tris-buffered saline with 0.1 % Tween-20 (TBS-T), the membrane was incubated overnight at 4 °C with specific primary antibodies for phospho-Smad1/5/8, total Smad, phospho-Erk1/2, total Erk1/2, phospho-p38, total p38 (Cell Signaling, Beverly, MA, USA) diluted 1:1,000 in 5 % skim milk in TBS-T. After washing, the blots were incubated for 2 h with anti-rabbit horseradish-peroxidase-conjugated antibody (Promega, Madison, WI, USA) diluted 1:3,000 in TBS-T. Signals were detected by an enhanced chemiluminescence reagent (Santa Cruz Biotech, CA, USA) and a LAS-4000 lumino-image analyzer system (Fujifilm, Tokyo, Japan).

Cells were harvested in a lysis buffer (Cell Signaling, Beverly, MA, USA) and then total protein concentration was determined by the BCA assay reagent (Bio-Rad Laboratories, Hercules, CA, USA). Proteins were resolved on a 10% SDS-PAGE and transferred to a PVDF membrane. After blocking in 5% skim milk in TBS with 0.1% Tween-20 (TBS-T), the membrane was incubated overnight at 4°C with primary antibodies for phospho-Smad1/5/8 and total Smad (Cell Signaling, Beverly, MA, USA) diluted 1:2,000 in 5% skim milk in TBS-T. After washing, the blots were incubated for 2 hrs with anti-rabbit horseradish-peroxidase-conjugated antibody (Promega, Madison, WI, USA) diluted 1:3,000 in TBS-T. Signals were detected by an enhanced chemiluminescence reagent (Santa Cruz Biotech, CA, USA), and LAS-4000 luminoimage analyzer system (Fujifilm, Tokyo, Japan).