Figure 1.
PRMT1 Interacts with PR in R5020-Stimulated T47D Breast Cancer Cells
(A) GST pull-down experiment: 35S-labeled in vitro translated PR-B, and ERα used as a positive control, were incubated with GST and GST-PRMT1 bound to glutathione Sepharose beads. The eluted proteins were analyzed by SDS-PAGE and visualized by autoradiography. Autoradiograph (upper) and Coomassie staining (lower) are shown.
(B) Whole-cell extracts (WCE) of T47D were subjected to immunoprecipitation (IP) using anti-PRMT1 antibody, or control IgG, and immunoblotted (IB) with anti-PR antibody.
(C) Proximity ligation assay (PLA) was used to detect the endogenous interaction between PRMT1 and PR in T47D cells, using anti-PR and anti-PRMT1 antibodies. T47D cells were transfected with control siRNA (siCT) or with anti-PRMT1 siRNAs (siPRMT1) and were cultured in medium deprived of steroids for 48 h, prior to the addition of R5020 (10 nM) for the indicated times. The nuclei were counterstained with DAPI (blue) (Obj: X60). The interactions are represented by red dots. Lower panel (left) shows the quantification of the number of signals per cell, as described in the Transparent Methods section. The mean ± SD of one experiment representative of three experiments is shown. The p value was determined using the Student's t test: ∗∗∗ indicates p ≤ 0.001. The efficacy of PRMT1 siRNA treatment is analyzed by IB and shown in the lower panel (right).