ROCK-II Activity Assay. Left: Active ROCK-II was incubated for 60 minutes at 30°C.
Right: 2.5 ng of active ROCK-II was incubated at 30°C for times as shown. Phosphorylation of
MYPT1 substrate was detected by anti-phospho-MYPT1 (Thr696) antibody as described in Assay Protocol.
ROCK-II Activity Immunoblot Assay. 25 μL of 1X Kinase Buffer containing 10 ng of
active ROCK-II was incubated with 50 μL of 1X Kinase Buffer containing 0.2 mM ATP and 500 ng of
recombinant MYPT1 for 30 minutes at 30°C. Kinase reaction was stopped by adding 25 μL of 4X
SDS-PAGE Sample Buffer. Lane 1, 3, 5, 7: Without kinase (negative control); Lane 2, 4, 6, 8: with
kinase. 200 ng (Lane 1 and 2), 100 ng (Lane 3 and 4), 50 ng (Lane 5 and 6) or 25 ng (Lane 7 and 8) of
recombinant MYPT1 substrate were loaded onto SDS-PAGE. Phosphorylation of MYPT1 substrate
was detected by anti-phospho-MYPT1 (Thr696) antibody as described in Assay Protocol.