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. Author manuscript; available in PMC: 2016 Dec 19.
Published in final edited form as: Biochemistry. 2015 Aug 3;54(32):5063–5071. doi: 10.1021/acs.biochem.5b00593

Figure 4. Effect of the R260S/G261L mutation on SPAK activity.

Figure 4

(A) Coupled protein kinase assay. SPAK 63–403 WT and R260S/G261L mutant were preincubated with ATP-MgCl2 for 30 minutes with or without WNK1, after which [γ-32P]-ATP was added for radiolabeling, and GST-NKCC2 1–175 was added as substrate. (B) Kinase assays comparing activity of SPAK 63–403 with and without the R260S/G261L mutation. T243E is an activating phosphomimetic mutation in the SPAK activation loop. All results in (A) and (B) are reported as relative activities, and as the mean and standard deviation of triplicate measurements quantified by phosphoimaging.