Materials and Methods

Cell Culture. AD human PCA LNCaP cells (ATCC) were cultured in RPMI 1640 containing 10% fetal bovine serum (FBS) and antibiotics, as described (14). Cells were transiently transfected using DMRIE-C reagent (In Vitrogen) and allowed to recover overnight. Cells were incubated in phenol red-free medium supplemented with 5% charcoal-stripped FBS (starvation medium) and exposed to agonists, as indicated. Where appropriate, cells were pretreated with inhibitors (H89, PD98059, 250 nM; U0126, 1 fiM) for 30 min prior to exposure to agonists.

ERK Activation Assay. Cells were incubated overnight in starvation medium, exposed to agonist for 5 min at 37°C, washed once with ice-cold phosphate buffered saline (PBS), and lysed in Laemmli sample buffer. Cell lysates were subjected to protein electrophoresis on 4-20% SDS-polyacrylamide gels and transferred onto nitrocellulose membranes. Phospho-ERK was detected using a 1:2,000 dilution ofa rabbit polyclonal phospho-ERK (Cell Signaling) specific antibody, and total ERK2 was detected using a 1:2,000 dilution of ERK2 (Cell Signaling) antibody. Blots were developed with a 1:7,000 dilution of horse-radish peroxidase-conjugated secondary antibody and specific protein bands visualized using enhanced chemiluminescence (Amersham).

Luciferase Assay. LNCaP cells transiently expressing luciferase gene fused to ARE-containing promoter (ARR3-luc/PB3) were grown in phenol red-free RPMI containing 2% charcoal-stripped serum overnight, and lysed in luciferase-lysis buffer (Promega). Protein concentrations were determined by the Bradford method. Relative luciferase units (RLU) were normalized to sample protein concentration. Results are presented as fold induction, the relative luciferase activity of transfected cells over the control cells.

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