The MC3T3-E1 cell line was a generous gift from Dr. Gideon Rodan (Merck, Sharpe and Dohme, West Point, PA). Purified human TGF-β1 was purchased from Upstate Biotechnology, Inc. (Lake Placid, NY). Recombinant human BMP-2 and rat heart Cx43 cDNA were generously provided by Dr. Gerard Reidel (Genetics Institute, Cambridge, MA) and Dr. David Paul (Harvard Medical School, Cambridge, MA), respectively. CT-360, a site-directed antibody to the C-terminal amino acids from positions 360 to 382 of the connexin 43 molecule derived from rat cardiac tissue, was generated in rabbit and was a most generous gift of Dr. Dale Laird (California Institute of Technology, Pasadena, CA).
MC3T3-E1 cells were plated at 5,000/cm2 on plastic 25 cm2 culture flasks or 10 cm2 round dishes (Costar, Cambridge, MA) or 25 mm2 round glass coverslips in α-MEM medium (Sigma, St. Louis, MO) supplemented with 5% fetal bovine serum, 26 mM NaHCO3, 2 mM glutamine, 100 units/ml penicillin, and 100 μg/ml streptomycin, and grown in humidified 5% C02/95% air at 37°C. Cells were passaged every 3–4 days after releasing with 0.002% pronase E in phosphate buffered saline (PBS). Cells were not used beyond passage number 40.
MC3T3-E1 cells in treatment groups were grown for 24 h, then incubated with BMP-2 (50 ng/ml) or TGF-β1 (2.0 ng/ml) dissolved in PBS containing 4 mM HCl and 0.1% bovine serum albumin (BSA) at 37°C for 24 and 48 h. Control groups received equal volumes of vehicle only (PBS with 4 mM HCl and 0.1% BSA).
Northern blot analysis
MC3T3-E1 total RNA was prepared using an acid guanidinium isothiocyanate-phenol-chloroform extraction method . Sample concentration was estimated by optical density (OD) at 260 nm. 20 μg of total RNA per lane were fractionated by electrophoresis on a formaldehyde-containing 1% agarose gel and transferred onto Duralon membranes (Stratagene, La Jolla, CA). Northern blots were hybridized with random primer radiolabeled (Megaprime, Amersham) rat heart Cx43 and GAPDH (control) cDNAs in Rapid-hyb buffer (Amersham Pharmacia Biotech, Piscataway, NJ) at 60°C overnight. The blots were washed at moderate stringency and autoradiographs were analyzed with densitometry.
Western blot analysis
MC3T3-E1 cell samples were lysed in 0.5 ml of RIPA buffer with slight modification (150 mM NaCl, 2 mM EDTA, 2 mM PMSF, 50 mM Tris-HCl, pH 7.4). 10 mM leupeptin and 2 mM sodium orthovanadate were added in the calf intestinal alkaline phosphatase (ALP) treatment groups. Cell lysates were sonicated (three 15 second pulses) and centrifuged at 4°C for 20 min. The protein concentration of each sample was determined by the Bradford method  using a BioRad protein assay kit (BioRad Laboratories, Hercules, CA). After alkalinization with NaOH to pH 9.7, one-half of each sample was treated with 1:500 diluted calf intestinal alkaline phosphatase (Sigma, St. Louis, MO) at 37°C for 3 h, and the other half was treated under the same conditions in the absence of ALP to demonstrate the phosphorylated isoform. The reaction was terminated by adding EDTA and heating the samples to 75°C for 10 min. 20 μg of total protein from each sample were separated using 10% SDS-polyacrylamide gel electrophoresis and transferred to Immobilon-P membranes (Millipore, Malborough, MA). The western blots were preincubated in blocking solution (5% nonfat milk, 0.2% Tween-20, 0.02% sodium azide in PBS, pH 7.4) at room temperature and probed with the primary anti-Cx43 polyclonal antibody (1:300 dilution; antibody against Cx43 C-terminus) for 1 h. The membranes were washed, incubated with the horseradish peroxidase-linked goat anti-rabbit IgG secondary antibody (Amersham), and developed using a chemiluminescence system (ECL kit, Amersham). Autoradiographs were analyzed with densitometry.
MC3T3-E1 cells were cultured on 22 mm2 glass coverslips and treated with BMP-2 or TGF-β1. The cells were fixed with 50% methanol/50% acetone at room temperature for 2 min, 1 ml of 0.1% Triton X-100 for 5 min, followed by three washes in PBS. The coverslips were incubated in blocking buffer for 45 min and probed with the anti-Cx43 polyclonal antibody (1:50 dilution) at room temperature for 1 h. Goat anti-rabbit IgG secondary antibody conjugated to DTAF was used to visualize the immunolocalized Cx43. The coverslips were mounted on glass slides using Permount medium (Fisher Scientific, Irvine, CA) and randomly viewed and imaged using epifluorescence microscopy with a blue-green filter set.
Fractionation and immunoprecipitation of Cx43
MC3T3-E1 cells were pre-incubated in methionine and cysteine-free culture medium for 45 min and labeled with 35S -methionine/35S-cysteine (ICN Biomedicals, Irvine, CA) at 37°C for 2 h. Cells were lysed in RIPA buffer as described above, incubated at 4°C for 10 min, vortexed, and collected in a microfuge tube on ice. The lysates were incubated and solubilized with 20% Triton X-100 at 4°C for 30 min and centrifuged. The membrane protein fraction was separated from the cytosolic fraction and reconstituted in RIPA buffer. Using the anti-Cx43 polyclonal antibody, Cx43 protein was immunoprecipitated, resolved with SDS-polyacrylamide gel electrophoresis (SDS-PAGE), and visualized by autoradiography.
Mean ± S.D. was calculated from each of two or more independent experiments. One way analysis of variance (ANOVA) followed by student's t test was used to determine significance (p < 0.05).