Immunofluorescence

PC3, SW480, HeLa, HEK293, MCF7, or BT474 cells were either transfected with the indicated plasmids or incubated with or without YM155. The cells were then fixed for 15 min in 4% paraformaldehyde at room temperature. After washing twice with PBS, cells were permeabilized in PBS containing 0.1% Triton for 5 min, washed extensively in PBS, and blocked with 3% bovine serum albumin (BSA). Cells were then incubated with the indicated primary antibodies diluted in BSA at 4 °C overnight. The next day, the cells were washed and incubated with appropriate Alexa Fluor 488/594-conjugated secondary antibodies (Invitrogen, CA, USA) for 1 h in the dark. Cells were washed three times with PBS in the dark and incubated with DAPI (Invitrogen) followed by mounting using VectaShield (Vector Laboratories, CA, USA) on glass slides. Cells were visualized and images were taken using a Leica fluorescence microscope (Leica, DM 5000B; Leica CTR 5000; Wetzlar, Germany).