Ppm1a

Western blot analyses were performed as previously described.^{13 (link)} Briefly, 30–50 µg total protein extract was run on a 10% SDS-PAGE gel, then transferred to a nitrocellulose membrane (GE Healthcare, Piscataway, NJ). Membranes were blocked in PBST using 5% non-fat milk or 5% BSA, then incubated overnight at 4 °C with PPM1A, Total and phospho-CDK2, total CDK4, total CDK6, phospho-RB, p21, and p27 antibodies (all from Cell Signaling Technology Inc., Danvers, MA, 1:1000), phospho-pan CDK antibody (Santa Cruz Bioscience, Dallas, TX, 1:500), phospho-CDK6 antibody (Thermo Scientific, Waltham, MA, 1:1000), total Rb antibody (BD Biosciences, San Jose, CA, 1:1000), or anti-Vinculin antibody (Sigma-Aldrich Corp., St. Louis, MO, 1:5000). Membranes were then washed and incubated with secondary anti-rabbit or anti-mouse antibody conjugated with IRDye (LI-COR, Lincoln, NE, 1:10,000) or anti-mouse/anti-rabbit HRP conjugated (GE Healthcare Bio-Sciences Corp., Piscataway, NJ, 1:3000). All blots shown derive from the same experiment, and were processed in parallel.

40μg of total protein was used for Western blot analysis. The samples were separated using 10–15% SDS–PAGE. These proteins were then transferred to polyvinylidene difluoride (PVDF, Millipore) membranes. After blocking with 5% non-fat milk, the PVDF membranes were incubated with primary antibodies in blocking buffer overnight at 4 °C. On the following day, PVDF membranes were incubated with the appropriate secondary antibodies for 2 hours at room temperature. After the membranes had been soaked in an enhanced chemiluminescence reagent (Thermo Scientific) for 5 minutes, the blots were visualized using X-ray film. Primary antibodies for PPM1A, anti-NICD1 and p-SMAD 1/5/8 (Cell Signaling Technology) were used in this analyses and β-ACTIN (sigma) antibody was used as a loading control. For the immunoprecipitation assay, 5 μg of either anti-NICD1 or Phospho-SMAD1/5/8 antibody was added to 500μg proteins extracted from control ATDC5 cells or NICD1 plasmid transfected cells in RIPA buffer (25 mM Tris-HCl, pH7.6, 150 mM NaCl, 1% Nonidet P-40, 1% sodium deoxycholate). Overnight incubation at 4 °C allowed complexes to form, after which 20 μl of 50% slurry protein A/G-agarose beads (Santa Cruz Biotechnology) was added and incubated at 4 °C for 3 hours. Immunoprecipitates were washed four times in RIPA buffer and analyzed by Western blots.