PC12/IA-2 cells treated with IA-2 siRNA followed by stimulation with low K+, high K+, or high K+ plus PMA decreased dopamine secretion by 45%, 38% and 22%, respectively, as compared to cells treated with control siRNA (Fig. influences the function of dense core vesicles and has a broad modulating effect on the cellular content and secretion of both hormones and neurotransmitters. for 10 min to remove insoluble debris. Protein concentrations in the supernatants were decided using Bradford protein assay (Bio-rad, Hercules, CA). The proteins in the extract (50 g) were separated on 10% SDS-PAGE. 2.5. Dopamine content Cells were seeded in 6-well culture plates at a density of 1 1 105 cells per well and cultured for 2 days. MEK162 (ARRY-438162, Binimetinib) The dopamine content was determined with a dopamine ELISA kit. 2.6. Dopamine secretion test PC12/mock and PC12/IA-2 cells were seeded in 6-well culture plates coated with poly-L-lysine at a density of 1 1 105 cells per well and cultured in DMEM for MEK162 (ARRY-438162, Binimetinib) 2 days. PC12 cells transfected with siRNAs also were incubated for 48 h after transfection. The dopamine secretion test was conducted as previously described MEK162 (ARRY-438162, Binimetinib) with slight modification (Shoji-Kasai et al., 2002). The cells were washed twice with a low (basal) K+ buffer (20 mM Hepes-NaOH, pH 7.4, 140 mM NaCl, 2.5 mM CaCl2, 1.2 mM MgSO4, 1.2 mM KH2PO4, and 11 mM glucose containing 4.7 mM KCl). After washing the cells were stimulated for 3 min with low K+ buffer (4.7 mM KCl) or with high K+ buffer (25 mM KCl) with or without 160 nM phorbol-12-myristate-13-acetate (PMA) (Sigma) for 6 min at 37 C. The media were collected and rapidly centrifuged at 10,000 for 20 s at 4 C. The supernatants then were measured for the amount of dopamine secreted. 2.7. Measurement of uptake and release of [H3]dopamine The experiment was performed as previously described (Ireland et al., 1995). PC12/mock and PC12/IA-2 cells were seeded in 6-well culture plates coated with poly-L-lysine at a density of 1 1 105 cells per well and cultured for 2 days. PC12 cells (1 105) transfected with siRNAs also were incubated for 48 h after transfection. Cells were washed three times with oxygenated KrebsCRinger bicarbonate (KRB) buffer made up of 118 mM NaCl, 4.7 mM KC1, 2.5 mM CaCl2, 1.2 mM KH2PO4, 1.2 mM MgSO4, and 24.2 mM NaHCO3 and was equilibrated with 5% CO2/95% O2. For the uptake assays, Rabbit Polyclonal to SNX3 [H3]dopamine (0.5 Ci) was added and incubated for 10 min at 37 C. After removal of excess radiolabeled ligands, the cells were washed rapidly three times with an ice-cold KRH buffer. The radioactivity remaining in the cells was extracted with NaOH and measured with liquid scintillation counter. For the release assays, cells were incubated with [H3]dopamine for 2 h and washed three times. The cells then were stimulated with low K+ buffer (4.7 mM KCl) or with high K+ buffer (25 mM KCl) or high K+ buffer plus 160 nM PMA for 6 min at 37 C. The media were collected and rapidly centrifuged at 10,000 for 20 s at 4 C. The radioactivity in the supernatants then was measured with a liquid scintillation counter. 2.8. Statistical analysis Each experiment was performed three times and assays were done in triplicate. Unless indicated otherwise, data are expressed as the mean standard error (SEM) of the three experiments. The Students test was used to determine statistical significance. 3. Results PC12 cells were transfected with full length human IA-2 (aa 1-979) and stable cell lines were established by G418 selection and limiting dilutions. Fig. 1a shows that the IA-2-transfected PC12.