Induction and localization of INPP4B in PC-3 cells. (A) Human prostate cancer cell lines were cultured in complete growth media, protein extracted and analyzed for INPP4B, PTEN and actin by Western blotting. (B) PC-3 clone #14 cells were cultured for 2 days in the presence or absence of 0.5 μg/ml doxycycline (Dox) in complete medium. Cells were fixed and stained with anti-FLAG M2 antibody, followed by staining with Alexa Fluor 488-conjugated rabbit anti-mouse secondary antibody. Nuclei were counterstained with DAPI. Scale bar = 20 μm (C) PC-3 clone #4 with moderate expression of INPP4B was cultured with increasing concentrations of doxycycline and the expression of FLAG-INPP4B and tubulin was analyzed by Western blotting. (D) Negative control, #4, and #14 clones were induced with 0.5 μg/ml Dox or left untreated. Protein lysates were analyzed for the levels of FLAG-INPP4B, phospho-Akt (S473), total Akt, and tubulin. (E) Using Carestream Molecular Imaging software, band intensities for pAkt 473 and total Akt were determined and p-Akt/Act ratios calculated in four independent experiments for clone #14. Values for pAkt/tAkt in untreated cells were assigned 100% and values for Dox treated cells adjusted proportionally. Percentage of remaining Akt activity in Dox treated cells was averaged for four independent experiments (p = 0.0191).