Phospho p44 42 mapk p erk1 2

Rat pheochromocytoma cells (PC12) stably transfected with rat M1 mAChR (PC12M1 cells) were seeded in 6-well plates at a density of 2×10⁶ cells/well. The following day, cells were washed twice with RPM1, and returned to the incubator with serum free media (starvation medium containing RPMI, 2mM Glutamine, 1% penicillin- streptomycin, 2.5µg/ml Amphotericin B, 0.1 mg/ml G418). On the next day cells were pretreated for 3 hours with AF710B at concentrations ranging from 0.1nM to 100nM, and then carbachol (10nM) was added for 10 minutes. In each plate, one well served as a control (no treatment) and one well as a positive reference in which the cells were treated with carbachol (10nM). Following this procedure, cells were collected and extracted with Ripa buffer (Sigma, R-0278, 200µl) containing a Protease inhibitor Cocktail (Sigma, 1:100) and Phenylmethylsulfonyl fluoride (Sigma, 1:1000). Phospho-p44/42 MAPK (p-ERK1/2) (1:1000, Cell signaling) and Phospho-CREB (ser 133) (1:1000, Cell signaling) were probed with anti-rabbit antibodies.

mMSCs were cultured on CS (control), SCS, CCS or BCS (N=6) for 7 days. The total protein from each group (40 µg) was fractionated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto a nitrocellulose membrane. The latter was blocked with 5% non-fat milk and incubated with primary antibodies against OPG (1:300, 11383, Santa Cruz Biotechnology Inc., Dallas, TX, USA), RANKL (1:300, 7628, Santa Cruz Biotechnology Inc.), β-actin (1:1000, 3700, Cell Signaling Technology, Danvers, MA,USA), Phospho-p44/42 MAPK (p-ERK1/2) (Thr202/Tyr204) (1:800, 4370, Cell Signaling Technology), total-ERK1/2 (1:1000, 4695, Cell Signaling Technology), Phospho-p38 MAPK (Thr180/Tyr182) (1:800, 4511, Cell Signaling Technology), total-p38 (1:1000, 9212, Cell Signaling Technology), Phospho-Akt (Thr308) (1:800, 4056, Cell Signaling Technology) and total-Akt (1:1000, 4691, Cell Signaling Technology). Signals were revealed after incubation with horseradish peroxidase-conjugated secondary antibody (1:5000, Santa Cruz Biotechnology Inc.) and enhanced chemiluminescence detection (GE Healthcare, Piscataway, NJ, USA). The stained bands were scanned and quantified using a densitometer (Syngene Bioimaging System; Frederick, MD, USA) and Scion Image software (Frederick). Protein expression levels were normalized against β-actin for each sample.

Cardiac microtissue protein was isolated after six days of culture following the manufacturer’s protocol for TRIzol Reagent (Invitrogen). Once the protein was isolated, the protein pellet was solubilized in a 1% SDS solution in de-ionized water. Proteins were separated by electrophoresis in Novex Tris-Glycine gels (Life technologies) and dry transferred using the iBlot (Life technologies) to a nitrocellulose iBlot Transfer Stack (Life technologies). Membranes were probed for Phospho-p44/42 MAPK (pERK1/2) (Cell Signaling Technology), p44/42 MAPK (ERK1/2) (Cell Signaling Technology), or GAPDH (Millipore) antibodies. Secondary antibodies were peroxidase conjugated (DAKO). Membranes were developed with ECL reagent LuminataClassico Substrate (Millipore).