The indicates what PAM-1 and PALm cell surface area levels would be if they were identical in scramble and sh-1A PAM-1 cells (= 3, **, < 0.01; *, < 0.05). Oregon Health and Science University or college using an Agilent 7700x equipped with an ASX 250 autosampler. The system was operated at a radio frequency power of 1550 watts, an argon plasma gas circulation rate of 15 liter/min, and argon carrier gas circulation rate of 1 1.08 liters/min. Elements were measured in kinetic energy discrimination mode using helium gas (4.2 ml/min). For measurement, samples were transferred to acid-treated 15-ml conical tubes. Data were quantified using a 9-point calibration standard (Common Elements Mix 2 Multi-Element Aqueous Standard, VHG Labs) in 1% HNO3. For each sample, data were acquired in triplicate and averaged. A Ge-72 internal standard (Internal Standard Multi-Element Mix 3, VHG Labs) launched with the sample was used to correct for plasma instabilities, and frequent measurements of a 10 ppb all-analyte answer as well as a blank (made up of 1% HNO3 only) were used as quality control and to determine the coefficient of variance. To assess recovery rates of elements and probe background contamination from containers, a certified NIST standard research material (Trace Elements in Water, 1643e) was digested and analyzed by the same method as the samples. HSG buffer was also analyzed to determine background contributions. Main Anterior Pituitary Cell Culture Rat anterior pituitary cells were plated on 0.16C0.19-mm-thick glass, 12-mm round coverslips (Fisher) coated with 0.1 mg/ml poly-l-lysine for 5 min followed by a rinse in NuSerum and two rinses in AtT-20 growth medium as explained previously (28). Briefly, rat anterior pituitary was rinsed in CSFM/air flow medium (DMEM/F-12 air flow, 25 mm HEPES, 100 models/ml penicillin, 100 g/ml streptomycin, 1C2 mg/ml BSA, ITS, 50 m ascorbate) and diced. Pituitary pieces were incubated in 0.75 ml of collagenase solution (4 mg/ml crude collagenase, 1 mg/ml hyaluronidase, 0.1 unit/ml benzonase, 10 mg/ml BSA) for 20 min at 37 C without CO2 under agitation. Pituitary fragments were diluted with 14 ml of CSFM/air flow and spun down at room heat for 5 min. Supernatant was removed, and cells were incubated with 0.75 ml of 3 mg/ml trypsin I-300 dissolved in CSFM/air for 5 min at 37 C without CO2 under agitation. Trypsin was blocked by adding 0.75 ml of 0.2 mg/ml lima bean trypsin inhibitor dissolved in AtT-20 medium. The dissociated cells were then filtered through a 70-m filter. After centrifugation of the flow-through, the cell pellet was resuspended in 5 ml of 160 mm NH4Cl to lyse reddish blood cells and Aloe-emodin then spun again for 5 min MLNR at room heat. The cell pellet was resuspended in 5 ml of AtT-20 growth medium. 1/25th of a rat anterior pituitary was plated per well of a 24-well dish. Cells remained in AtT-20 growth medium for 2 days and were then switched to DMEM/F-12, 25 mm HEPES, 100 models/ml penicillin, 100 g/ml streptomycin, 1C2 mg/ml BSA, ITS, 50 m ascorbate. Cells were used on days 3 and 4. Manipulation of Cells Cells were incubated in DMEM/F-12 air flow medium made up of 25 mm HEPES, pH 7.4, 1 mg/ml BSA for 30 min at 37 C without CO2. For transferrin uptake experiments, cells were then incubated in DMEM/F-12 air flow medium made up of 25 mm HEPES, pH 7.4, 1 mg/ml BSA, 25 g/ml Alexa Aloe-emodin Fluor 546 transferrin (Life Technologies, Inc.) for 10 min at 37 C without CO2. Cells were fixed using 4% formaldehyde in PBS for 20 min at room heat. For nocodazole treatment experiments, cells were incubated for 10 min at 37 C in DMEM/F-12 air flow medium made up of 25 mm HEPES, pH 7.4, 1 mg/ml BSA, and 10 m nocodazole (Sigma) or the equivalent volume of DMSO followed by 10 min in the same medium containing 25 g/ml Alexa Fluor 546 transferrin. BCS and Copper Treatment of AtT-20 Cells Cells were first incubated for 30 min in DMEM/F-12 medium or DMEM/F-12 air flow medium containing ITS, 25 mm HEPES, pH 7.4, 1 mg/ml BSA. Cells were equilibrated in this medium during two consecutive 30 min incubations at 37 C with 5% CO2 (DMEM/F-12 medium) or without CO2 (DMEM/F-12 air flow medium). Cells were then treated with medium made up of 50 m bathocuproinedisulfonic acid (BCS, Sigma) Aloe-emodin overnight or CuCl2 (20 or 200 m, Sigma) for 2 h or medium only as a control. Cells were either fixed for immunostaining, lysed in detergent for biochemical analysis, or chilled for cell surface biotinylation. Using an antibody against the C terminus of Atp7a (17), we observed a 2-fold increase in transmission when AtT-20 cells were exposed to 20 m copper for 2 h. The inability of cycloheximide, a protease synthesis inhibitor, to block this increase and the failure of an N-terminally directed Atp7a antibody to detect the increase suggested the occurrence of a copper-responsive post-translational modification. Detection of JP Amidation Antibody specific to the amidated C.