Figure 3. A screen to identify kinase inhibitors that antagonize KCC3 Thr⁹⁹¹/Thr¹⁰⁴⁸ phosphorylation.

(A) Results from the primary kinase inhibitor screen. We performed a screen to identify kinase inhibitors that decreased KCC3 P-Thr⁹⁹¹ using a library of >220 well-characterized, cell-permeable protein kinase inhibitors (see Methods). Dox-induced HEK293-KCC3 WT cells were exposed to 20 μM of kinase inhibitor for 2 hours in 24-well plates. Lysates were harvested, subjected to SDS-PAGE, and Western blot with the indicated antibodies. Quantitative measurement of the KCC3 P-Thr⁹⁹¹: MYC signal ratio (MYC) was performed as described in Methods. Top hits from the screen are listed below a representative blot from the screen. Drugs showed overlap under several different signaling pathways listed in different colors. (B) Results from the secondary kinase inhibitor screen. We tested representative drugs targeting pathways that scored positively in the primary screen at lower concentrations and with decreased incubation times (1 μM for 30 min) to promote inhibitor specificity for the intended target kinase. HEK293 cells expressing N-terminal FLAG epitope tagged KCC3 were treated 30 min with isotonic low Cl⁻ and hypotonic low Cl⁻ conditions, then treated in the same conditions with the indicated inhibitor concentrations for an additional 30 min. (C) Concentration-response experiments of STOCK1S-50699 on KCC3 P-Thr⁹⁹¹/Thr¹⁰⁴⁸. HEK293 cells were transfected with DNA construct encoding wild type N-terminal FLAG-tagged KCC3. 36 h post-transfection, cells were exposed 30 min to either control isotonic conditions or hypotonic low Cl⁻ conditions, then treated in the same conditions with STOCK1S-50699 at the indicated concentrations for an additional 30 min. Lysates were and subjected to SDS-PAGE and Western blotting with the indicated antibodies (lower panel). (D) Quantitation of immunoblot data from (C) presented as ratios of phospho-KCC3/total KCC3. ***p < 0.001, **p < 0.01, *p < 0.05, ns: non-significant.