Figure 3
Two-Dimensional separation of ROCK2-phosphorylated HEK293 cellular homogenate. A. HEK 293 cells were transfected with either W1161A or M160A/W1161A ROCK2. The following day, ROCK2 was immunoprecipitated and resuspended in HEK 293 cellular homogenate (60 μg) with γ32P-N6(Benzyl)ATP in activation buffer for 30 minutes at 30°C. A small sample was taken and separated by SDS-PAGE followed by staining with Coomassie Blue and exposure to film. B. The remaining sample of M160A/W1161A ROCK2-phosphorylated homogenate was separated from the beads and proteins were precipitated with acetic acid/TCA, and then resuspended in 2D sample buffer. Proteins were loaded onto pH 3-11NL or pH 4-7 L IEF strips, as indicated. Following overnight IEF, the samples were separated in the 2nd dimension by 8% SDS-PAGE. Gels were then silver-stained, dried (upper panel), and exposed to Amersham Hyperfilm (lower panel). Dashed circles indicate putative ROCK2 substrates that were excised and sent for identification. Results are representative of three independent experiments.