Figure 3. DHEA binds with high affinity to HEK293^TrkA and HEK293^p75NTR cell membranes.

(A, C) [³H]-DHEA saturation binding assays and Scatchard blots in HEK293 cells, transfected with the plasmid cDNAs of TrkA and p75^NTR receptors. Fifty µl of cell membrane suspension in triplicate were incubated for 30 min at 25°C with 1–30 nM [³H]-DHEA in the presence or absence of 500-fold molar excess of DHEA. Western blot inserts show the efficacy of transfection (K_D represents the mean ± SE of 3 experiments) (E) [³H]-DHEA saturation binding assays in PC12 cells, transfected with the si/shRNAs against TrkA and p75^NTR receptors or with the siRNA against GAPDH. The efficacy of transfection is shown in Western blot inserts and in FACS analysis (F), (K_D represents the mean ± SE of three experiments). (B, D) Fluorescence localization of DHEA membrane binding on HEK293 cells transfected with the plasmid cDNAs of TrkA and p75^NTR receptors. Transfectants were incubated with either the membrane impermeable DHEA-BSA-FITC conjugate (100 nM), BSA-FITC (100 nM), or with specific antibodies against TrkA and p75^NTR proteins. Transfectants were analyzed under the confocal laser scanning microscope (B) or by FACS analysis (C). Blue staining depicts Hoechst nuclear staining.