Recombinant RSK2 was expressed, purified, and activated according to our established protocol (69), with an additional purification step using Strep-Tactin resin before final size exclusion. RLC20 (100 μM) purified from turkey gizzard was incubated with either activated RSK2 (0.12 μM) or MLCK (0.28 μM) purified from turkey gizzard, serving as a positive control, for 10 min at 30°C in buffer containing 25 mM Mops-NaOH (pH 7.0), 10 mM MgCl2, 1 mM EGTA, 5% glycerol, 1 μM microcystin-LR (MCLR), 1 mM adenosine triphosphate (ATP), 4 mM Pefabloc, and 0.5 mM tris (2-carboxyethyl) phosphine hydrochloride (TCEP). Sample buffer was added to the reaction mix, and samples were heated to 100°C for 5 min and subjected to electrophoresis and Western blotting. RLC20 phosphorylation was expressed as the ratio of RLC Ser19 phosphorylation to total RLC protein.

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