Fig. 3
Nuclear PRDX5 binds to the BRCA2 silencer in dividing cells. a An autoradiogram from DNA affinity purification using 35S-methionine-labeled nuclear extracts of quiescent (Q) and dividing (D) BT549 cells to detect proteins that bind to the BRCA2 silencer. Band A (~ 29 kDa) corresponds to SLUG and band B (~ 18 kDa) corresponds to PRDX5. b A supershift assay showing PRDX5 binding to the BRCA2 silencer. The 5′-biotynylated silencer DNA (221-bp) served as a probe. The probe was incubated with unlabeled nuclear extract (NE) from dividing BT549 cells. PRDX5 antibody was used to verify PRDX5 binding to the silencer probe. c Validation of the expression of C-terminal FLAG-tagged PRDX5A in BT549 cells. Transient transfection was performed using either the vector (p3XFLAG-CMV14) alone or p3XFLAG-CMV14-PRDX5A for 48 h before the isolation of the cytosolic (CF) and nuclear (NF) fractions. Fibrillarin, HSP90, and GSK3β served as a nuclear marker, a cytosolic marker, and the loading control, respectively. d Immunofluorescence analysis with anti-FLAG antibody (green) showing puncta of C-terminal FLAG-tagged PRDX5A in the nucleus and cytoplasm of the transiently transfected cells. The nuclei were stained blue with DAPI. e A ChIP assay showing in-vivo binding of PRDX5A at the silencer in breast cells transiently expressing FLAG-tagged PRDX5A. Anti-FLAG antibody was used to pulldown the protein. Anti-mouse IgG was used as a control antibody. The lower panel shows the amplification of input DNA prior to immunoprecipitation