Fig. 6. Inhibition of Hsp90 does not affect EGFP-Rab3A dynamics or exocytosis in PC12 cells. (A) Cells were transfected with 0.5 µg EGFP-Rab3A and at 18 hours post transfection small regions of interest (ROIs) in each cell were bleached with high intensity laser. Fluorescence recovery was recorded over time and corrected to account for bleaching sustained during low-intensity imaging, summed and normalised to the first data point post-bleach. Data are shown from control cells and cells recorded in parallel that had been treated with 10 µM geldanamycin (GA) for 1 hour before bleaching (n=11 for each condition). (B) Secretion of exogenous human growth hormone (hGH) from cells treated with varying concentrations of GA for 1 hour before stimulation was assayed. Stimulated cells were exposed to 300 µM ATP, hGH release over 15 minutes was assayed and expressed as a percentage of total hGH. (C) Representative image of control cells expressing glucocorticoid receptor-EGFP (GCR-EGFP) 18 hours after transfection with 0.5 µg GCR-EGFP showing two adjacent cells in which the GCR-EGFP is either predominantly cytosolic or alternatively nuclear. Bar, 2 µm. (D) Effect of geldanamycin on GCR-EGFP translocation to the nucleus. After transfection with GCR-EGFP, treated cells were exposed to 1 µM dexamethasone on ice for 1 hour or 1 µM dexamethasone on ice for 1 hour with the addition of 10 µM geldanamycin for the final 30 minutes. Following a 20-minute incubation at 37°C and fixation, 100 cells were scored for each condition according to the cytosolic or nuclear localisation of GCR-EGFP Data are expressed as cytoplasmic:nuclear ratios and are representative of two independent experiments.