Fig. 2

AKAP13 is required for PKA-induced ERαS305 phosphorylation and interacts with ERα and PKA-RII. a siRNA targeting AKAP13 prevents PKA-induced ERαS305 phosphorylation. MCF-7 breast cancer cells were transfected with an siRNA targeting AKAP13 or a control siRNA, after which the cells were treated for 1 h with 10 μM forskolin or left untreated. Samples were analysed by SDS-PAGE and Westernblotting, probing with antibodies detecting AKAP13, ERαS305P, ERα or actin as a loading control. b AKAP13 is required for a forskolin-enhanced ERα/PKA-cat interaction. Estrogen Receptor-negative U2OS cells were transfected with CFP-tagged ERα, YFP-tagged PKA catalytic subunit and a non-tagged PKA regulatory RII subunit. In addition, cells were transfected with an siRNA targeting AKAP13 or a control siRNA. Energy transfer from the CFP to the YFP fluorophore was measured in the same cell before and after 1 h of 10 μM forskolin treatment, and the average value prior to treatment was set on 1. N > 10. Bars indicate SEM. A student’s T-test was performed; p < 0.05. c, d, e AKAP13, ERα and PKA-RII form a complex. MCF-7 cells were hormone deprived for 3 days to deplete activated ERα transcriptional processes. Following that, cells were lysed for immunoprecipitations, directed at PKA-RII (c), AKAP13 (d), ERα (e) or a negative control protein. In addition, input and supernatant (sup) samples were taken. Western blots were probed with antibodies detecting AKAP13, ERα and PKA-RII