Fig 1: Subcellular distribution of HA-PDRG1 and the HA-PDRG1/ MATa1 interaction.(A) Representative western blots of nuclear (N) and cytoplasmic (C) fractions (50–70 µg) obtained from HEK 293T cells transiently transfected with pHA or pHA-PDRG1 in three independent experiments. Membranes were analyzed using anti-HA, anti-TBP (nuclear marker) and anti-tubulin (cytoplasmic marker). (B) Representative confocal immunofluorescence images (N = 50 per cell line) of CHO, COS-7, HEK 293T and H35 cells transiently cotransfected with pHA-PDRG1 and pFLAG-MAT obtained using mouse anti-HA, rabbit anti-MATa1 and the corresponding secondary antibodies coupled to Alexa Fluor 488 (green) or 546 (red). Colocalization of both proteins is shown in yellow (scale bar = 12 µm) (C) Representative results of anti-FLAG immunoprecipitations carried out in N and C fractions obtained from HEK 293T cells transiently cotransfected with pFLAG-MAT and pHA-PDRG1 or the empty plasmids (mock). Input fractions were analyzed using anti-FLAG, anti-HA, and antibodies against nuclear and cytoplasmic markers, whereas immunoprecipitates were examined using anti-HA. Results correspond to a minimum of three independent experiments. (D) Representative confocal immunofluorescence images of nuclear matrix preparations of HEK 293T and CHO cells overexpressing HA-PDRG1 using rat anti-HA (green), mouse anti-SC-35 (red) and appropriate secondary antibodies conjugated to Alexa Fluor dyes (N = 50 per cell line); colocalization appears in orange. Scale bar = 12 µm.