Fig. 3.

Calcineurin (CaN) stabilizes ER-α through dephosphorylation at Ser²⁹⁴. (A) Phosphatase assay performed with immunoprecipitated, FLAG-tagged ER-α with or without recombinant CaN and calmodulin. The reaction mixtures were subjected to immunoblot analysis with antibodies to Ser²⁹⁴-phosphorylated ER-α and to FLAG. The relative phosphorylated ER-α/FLAG band intensity ratio was determined as the mean ± SEM from three independent experiments. **P < 0.01 (paired t test). (B) MCF7 cells were treated with 4 μM A23187 for the indicated times, after which total cell lysates were subjected to immunoblot analysis with the indicated antibodies. The relative ER-α/β-actin band intensity ratio was determined as mean ± SEM values from four independent experiments. **P < 0.01 and ***P < 0.001 versus the corresponding value for time 0 (Dunnett’s test). (C) MCF7 cells were transiently transfected with empty vector or expression vectors for FLAG-tagged S294A mutant or WT forms of ER-α for 2 d, after which cell lysates were subjected to immunoblot analysis with antibodies to ER-α. The relative ER-α/β-actin band intensity ratio was determined as mean ± SEM values from five independent experiments. P < 0.05 (paired t test). (D) HEK293T cells were transiently transfected with expression vectors for V5-tagged WT or S294A mutant forms of ER-α for 2 d, after which cell lysates were subjected to immunoprecipitation (IP) with antibodies to V5. The resulting immunoprecipitants as well as a portion of the original cell lysates (Input) were subjected to immunoblot analysis with antibodies to ubiquitin (Ub), to E6AP, and to V5. The relative Ub/V5 and E6AP/V5 band intensity ratios were determined as the mean ± SEM from three independent experiments. P < 0.05 (paired t test).