Figure 5.

The differences between the hGR wild type and D196Y mutant for phosphorylation state and binding to NCoR. (a) Phos-tag PAGE for phosphorylation of EGFP-hGR^WT and EGFP-hGR^D196Y. In the absence of Dex (lane 1–4), any differences in the band-shifts were not observed; (b) the enlarged image of lane 5–8 of (a) after stimulation with Dex. The band of EGFP-hGR^WT (from lane 5 to 6) and EGFP-hGR^D196Y (from lane 7 to 8) was shifted by the addition of the phosphatase. However, the band shift caused by the dephosphorylation of EGFP-hGR^D196Y, corresponding to the shift from (i) to (iii), was greater than that of EGFP-hGR^WT, corresponding the shift from (ii) to (iii); (c) binding of NCoR with hGR wild type and D196Y mutant was analyzed by coimmunoprecipitation. The tendencies of binding NCoR with the hGR wild type and D196Y mutant were shown as normalized to the relative intensity [I_NCoR/I_GR] of the bands quantified from Figure S5a,b. The mean and SD of normalized relative intensity [I_NCoR/I_GR] was obtained from three individual experiments. *: p < 0.05, ns: no significant differences. (Student t-test).