Figure 2. Regulation of ERα/PI3K and ERα/Src interactions in MCF-7 cells detected by PLA.

1. MCF-7 cells transfected with control siRNA duplexes or with specific PRMT1 siRNA duplexes were controlled for PRMT1 expression by Western blot.
2. We analysed by PLA ERα/PI3K (panels a,b) and ERα/Src dimers (panels c,d).
3. Quantification of the number of signals was performed as described. The mean ± SEM of four experiments is shown. p-value was determined by Student's t-test.
4. MCF-7 cells, treated or not with PP1 (5 µM) or LY294002 (20 µM) 15 min before E₂ treatment, were incubated with the vehicle or with E₂ for 5 min. Cell lysates were immunoprecipitated with anti-Src and blotted with indicated antibodies.
5. ERα/PI3K (panels a–c) and ERα/Src (panels d–f) interactions were analysed by PLA in cells treated as described above.
6. Quantification of the number of signals was performed for each couple as described. The mean ± SEM of four experiments is shown. p-value was determined by Student's t-test.
7. MCF-7 cells were incubated with 1 nM of a peptide mimicking hERα 536–541 containing Y537 (Y-pep) or the corresponding phosphorylated peptide (pY-pep) 30 min before E₂ treatment. Then, cell lysates were immunoprecipitated with anti-Src and blotted with indicated antibodies.
8. From the same experiment, ERα/PI3K (panels a,b) and ERα/Src interactions (panels c,d) were analysed by PLA.
9. Quantification of the number of signals was performed as described. The mean ± SEM of four experiments is shown. p-value was determined by Student's t-test.