Fig. 2

ERα-36 interacts with PR. a A radioactive GST pull-down assay was performed by incubating in vitro ³⁵S-labeled PR (PR #) with GST, GST-ERα-36, and GST-ERα-36ΔC. The corresponding Coomassie-stained gel is shown below. Arrows indicate the full-length fusion proteins. b PR was divided into 5 fragments (PR1 to PR5). Radioactive ERα-36 (ERα-36 #) was incubated with the different domains of PR fused to GST, and the bound proteins were visualized by autoradiography. The corresponding Coomassie-stained gel is shown below. “>” indicates the full-length fusion proteins. c pSG5Flag-ERα-36 and pCDNA3V5-PR were overexpressed in Cos7 cells. Cell lysates were immunoprecipitated with the anti-Flag antibody, and the presence of ERα-36 and PR was visualized by Western blot using the anti-Flag and anti-V5 antibodies, respectively. The lower panel shows the expression of the different proteins in the input. d Proximity ligation assay (PLA) was used to detect the cellular co-localization of endogenous ERα-36 and PR in T47D, grown on coverslips in 12-well plates. Cells were transfected with control siRNA (siCtl) or with siRNA against PR (siPR) and treated for the indicated times with 10 nM of R5020. PLA for ERα-36/PR interaction was performed with anti-PR- and anti-ERα-36-specific antibodies. The nuclei were counterstained with DAPI (blue) (Obj, × 60). The detected interactions are represented by red dots. e Quantification of the number of signals per cell was performed using computer-assisted analysis, as reported in the “Methods” section. The mean ± SD of one experiment representative of three experiments is shown. f The efficacy of PR siRNA treatment analyzed by Western blot analysis is shown in the left-hand panel and quantified in the right-hand panel where the PR expression relative to tubulin was quantified using ChemiDoc MP (Biorad)