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. 2010 Sep 22;24(11):2126–2138. doi: 10.1210/me.2010-0170

Figure 3.

Figure 3

Acetylation-deficient PR displays delayed phosphorylation. A, Stable expression of acetylation-deficient PR in breast cancer cells. T47D cells stably expressing wt or K-A PR-B (clone nos. 101, 102, and 111) were Western blotted for total PR and β-actin. B, K-A PR-B exhibits altered SDS-PAGE upshift. T47D cells stably expressing K-A or wt PR-B were serum starved for 24 h and treated for 0, 10, or 60 min with R5020 (10−8 m). Cell lysates were Western blotted for total PR and β-actin as a loading control. Data correspond to images from the same SDS-PAGE and film exposure. C, Acetylation-deficient PR displays a lag in phosphorylation on specific sites. T47D cells stably expressing wt or K-A PR-B were serum starved for 24 h and treated for 0, 30, or 180 min with R5020 (10−8 m). Cell lysates were Western blotted with phospho-specific antibodies against Ser190, Ser294, Ser345, and Ser400, as well as total PR. D, K-A PR-B exhibits less Ser400 phosphorylation compared with wt PR-B. Triplicate cultures of HeLa cells were transfected with wt or K-A PR-B, serum starved, and treated for 1 h with vehicle or R5020 (10−8 m). Lysates were Western blotted with antibodies against total PR and phospho-Ser400 PR. Total PR (red) and phospho-Ser400 PR (green) band intensities were quantified using an Odyssey Imaging System. Phosphorylation was normalized to total PR levels (±sd; *, P < 0.005; **, P < 0.001). Each experiment was performed three to four times. EtOH, Ethanol.