PC12 cells have both intracellular and membrane ERβ. Monoclonal Ab clone 9.88 was used for detection. A. Protein levels of ERβ in whole-cell extracts were determined by immunoblot analysis. ERβ is present in cells grown in serum-free medium, but is not induced by NGFβ over a 7-day period. Representative of 3 experiments. B. Immunocytochemistry of non-permeabilized cells treated with 20 ng/ml NGFβ for 2 days before staining. The bars represent 2 μm. Fluorescent image micrographs were viewed with an FITC filter were photographed. Left panel: Transmission image of middle panel. Middle panel: membrane ERβ immunofluorescence present heterogeneously on cells. Right panel: Punctate ERβ staining is irregularly distributed on the cell surface of a more highly magnified cell. C. Both nonpermeabilized cells (symbols) and permeabilized cells (bars) were assessed for ERβ by the plate assay over an Ab saturation curve. Controls (C) are as previously described in Figures 1 and 3. The cross-hatched bar is clathrin from permeabilized cells; ▼ = clathrin signal from unpermeabilized cells. The solid gray bar represents combined controls (IgGκ, no1°, no2° Abs) from permeabillzed cells and ▲ = the same combined controls in unpermeabilized cells. The white bars represent values for ERβ Ab 9.88 detection in permeabilized cells at the concentrations shown on the X axis. This graph represents average values from 2 experiments (each with multiple samples) ± S.E.M.