Immunofluorescence was used to evaluate the localization of NRF2 in the presence of PML/RARa. U937-PR9 and Mock control cells were prepared using a cytocentrifuge. Assays were performed as previously described [35 (link)]. Briefly, cells fixed with 4% paraformaldehyde (PFA) were permeabilized in PBS containing 0.1% Nonidet P-40 and blocked in 3% bovine serum albumin (BSA) (Sigma-Aldrich, St. Louis, MO, USA). Slides were incubated overnight with the primary antibody anti-PML, kindly provided by Brunangelo Falini; and anti-NRF2 (Ab Cam, Cambridge, UK); and following two PBS washes and incubated for 2 h with the secondary antibodies: Alexa Fluor 555-labeled goat anti-mouse and Alexa Fluor 488-labeled goat anti-rabbit (diluted 1:400 with PBS+BSA 3%) (Invitrogen, Carlsbad, CA, USA).
The nuclei were stained with 1 μg/mL DAPI (4′,6-diamidino-2-phenylindole) (Sigma-Aldrich, St. Louis, MO, USA) for 5 min in PBS. Finally, cells were rinsed and mounted in Fluoromount (Sigma-Aldrich). Images were acquired using a Zeiss LSM 700 (Carl Zeiss Microscopy, Jena, Germany) confocal laser scanning microscope.
The nuclei were stained with 1 μg/mL DAPI (4′,6-diamidino-2-phenylindole) (Sigma-Aldrich, St. Louis, MO, USA) for 5 min in PBS. Finally, cells were rinsed and mounted in Fluoromount (Sigma-Aldrich). Images were acquired using a Zeiss LSM 700 (Carl Zeiss Microscopy, Jena, Germany) confocal laser scanning microscope.
Free full text: Click here
