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Total protein was extracted from cells following transfection using Laemmli buffer and subjected to western blot analysis as described earlier.²⁹, ³⁰ After electrophoresis, the separated proteins were transferred to PVDF membrane using wet method. The membranes were incubated with rabbit polyclonal antibody to USF‐1 (SC‐229; Santa Cruz Biotechnology). The bound antigen–antibody complex was detected by HRP conjugated secondary antibody (Santa Cruz Biotechnology) and the electrochemiluminescence plus western blot detection system (GE Healthcare UK Limited). The same membrane was used for β‐actin levels detected by anti‐β‐actin antibody (Sigma Chemical Company) as an internal loading control.

PC‐3 cells were cultured on Lab‐Tek chamber slides (Nunc Inc.) and 24 h after various treatments were fixed in buffered formalin. Non‐specific sites were blocked with 3% bovine serum albumin (BSA) in phosphate buffered saline (PBS) (pH 7.4) with 0.25% Tween 20 for 30 min. Slides were then incubated overnight at 4°C in primary rabbit polyclonal antibody to USF‐1 (SC‐229; Santa Cruz Biotechnology) that was diluted to 1:100 with the blocking buffer. After washes in PBS (pH 7.4) with 0.25% Tween 20 (3 times each for 15 min), the cells were exposed to secondary antibody, FITC‐conjugated anti‐rabbit IgG that was diluted to 1:1000 in blocking buffer. After three washes in PBS (pH 7.4) with 0.25% Tween 20 (3 times each for 15 min) and one wash in PBS for 15 min, the slides were mounted with aqueous mounting media using antifade and DAPI (4′,6‐diamidino‐2‐phenylindole) (VectaShield, Vector) and visualized using triple band pass filter in Nikon epifluorescence microscope.