Figure 3.

Representative figure of primary screening of SPAK inhibitors in the newly developed ELISA system. Each compound is added in the kinase reaction at a final concentration of 50 μM. As a positive control indicating nonphosphorylation of NKCC2, ATP-free kinase buffer is used. A kinase reaction without any compounds is used as a negative control for screening. The arbitrary cut-off value of OD₆₂₀ is set to 0.2. The compound shown as a white bar is regarded as a positive compound having a strong inhibitory effect on SPAK-regulated NKCC2 phosphorylation.