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. 2017 Nov 1;292(52):21253–21263. doi: 10.1074/jbc.M117.817841

Figure 6.

Figure 6.

NEM decreases KCC2 Thr-1007 phosphorylation levels. A, validation of KCC2 pThr-1007 antibody in KCC2-transfected HEK293 cells. Both KCC2-WT and KCC2-T1007A constructs were expressed in HEK293 cells. Immunoprecipitated KCC2 was probed for both pThr-1007 and total KCC2. The loss of pThr-1007 signal with expression of the KCC2-T1007A construct demonstrates the specificity of this antibody for the phosphorylated state of Thr-1007. B, KCC2 was immunoprecipitated from untransfected (U) or DMSO-treated or NEM-treated KCC2-transfected HEK293 cell lysate. Immunoprecipitated KCC2 was probed for both total KCC2 and KCC2 pThr-1007. KCC2 pThr-1007 levels were quantified relative to the DMSO control (n = 3; unpaired t test, p = 0.0007). Error bars represent S.E. C, KCC2 was immunoprecipitated from DMSO-treated or NEM-treated immature cortical neuron lysate. Immunoprecipitated KCC2 was probed for both total KCC2 and KCC2 pThr-1007. KCC2 pThr-1007 levels were quantified relative to the DMSO control (n = 3; unpaired t test, p < 0.0001). Error bars represent S.E. ***, p < 0.001; ****, p < 0.0001.