Western blot
Cells are solubilized in hot 2× SDS gel sample buffer (20 mM dithiothreitol, 6% SDS, 0.25 M Tris, pH 6.8, 10% glycerol, 10 mM NaF and bromophenyl blue) at 2 × 106 per mL. The extracts are heated in a boiling water bath for 5 minutes and sonicated with a probe sonicator with 3–4 bursts of 5–10 seconds each. Samples are diluted with 1× SDS sample buffer to the desired loading of 1–5 × 103 per lane. Lysates were resolved by SDS-PAGE, transferred to Immobilon-P membrane, and immunoblotted with 0.5 μg/mL primary Abs as described in R&D Systems Website [15].