The assays for MLCP were conducted as described previously (69). Briefly, thiophosphorylation of recombinant MLCP complex (0.6 μg: FLAG/MAT/Venus-MYPT1 + 3xHA-PP1δ) was carried out for 2 hours at 30°C with RSK2 (12 mU/μl; BioVision) or ROCK2 (27 mU/μl; SignalChem) in the presence of 0.1 mM ATPγS and terminated by adding 2 μM staurosporine. Aliquots of the thiophosphorylated MLCP were mixed with 2 μM P-RLC20 (3–20) peptide as substrate. After 60-min incubation, at room temperature, phosphate released from the substrate was assayed using the Malachite green method (BioMol). The MLCP activity without ATPγS was set as 100%. RSK2 activity was confirmed by phospho-dot blotting using RLC20 as substrate.

Note: The content above has been extracted from a research article, so it may not display correctly.

Please log in to submit your questions online.
Your question will be posted on the Bio-101 website. We will send your questions to the authors of this protocol and Bio-protocol community members who are experienced with this method. you will be informed using the email address associated with your Bio-protocol account.

We use cookies on this site to enhance your user experience. By using our website, you are agreeing to allow the storage of cookies on your computer.