Figure 8

Nuclear translocation of NFκB in HRPC. The HRPC cells were cultured with normoxia/hypoxia HUVEC-CM for 24 hr and subcellular localization were done with immunofluorence and Western blot study. (A) HRPC cells were cultured with normoxia HUVEC-CM and intracellular location of NFκB was determined by immunofluorescence using primary anti-NFκB antibody and secondary antibody FITC labeled IgG. Nuclei were counterstained with DAPI. The immunofluorescence images observed in higher magnification showed that NFκB was more localized to the cytoplasm and less to the nuclei of the HRPC cells. (B) HRPC cells were cultured with hypoxia HUVEC-CM and labeled with anti-NFκB and nucleus staining with DAPI (blue). Hypoxia HUVEC-CM activates NFκB and translocated the most of the NFκB protein in the nuclear region of the HRPC cells. (C) Protein were extracted from the nucleus and the cytoplamic fraction of the HRPC cells after the cells were exposed with normoxia/hypoxia HUVEC-CM for 24 hr. 20 μg of protein from nuclear extracts and cytosol were separated by SDS-PAGE followed by western blotting with primary anti-NFκB antibody and followed by the secondary antibody HRP-conjugated. (D) Densitometry quantification of signal intensities of NFκB and β-actin was performed using a Bio-Rad scanning densitometer and Quantity One analysis software. Results are in arbitrary units and expressed as mean +/- SE of three independent experiments.