On coverslips pre-coated with poly-L-lysine in 6-well plates and transfected with

On coverslips pre-coated with poly-L-lysine in 6-well plates and transfected with 100 ng of GFP-tagged receptors. For co-localization of GFPtagged receptors with the ER marker DsRed2-ER, HEK293 cells grown on coverslips were transfected with 100 ng of GFP-tagged receptors and 100 ng of pDsRed2-ER. The cells were fixed with 4 paraformaldehyde-4 sucrose mixture in PBS for 15 min and the coverslips were mounted with prolong antifade reagent containing DAPI. Images were captured using a Zessis confocal microscope (LSM510) equipped with a 63x objective (NA = 1.3). The colocalization of the receptor with the ER marker DsRed2ER was determined by Pearson’s coefficient 12926553 using the ImageJ JaCoP plug-in as described [46].Cell Culture and Transient TransfectionHEK293 and HeLa cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) with 10 fetal bovine serum (FBS), 100 units/ml penicillin, and 100 units/ml streptomycin and transiently transfected by using Lipofectamine 2000 reagent as described previously [44]. Transfection efficiency was estimated to be greater than 70 based on the GFP fluorescence.Measurement of ERK1/2 ActivationHEK293 cells were cultured in 6-well dishes and transfected with 1 mg of a2A-AR or its mutants as described above. After 24 h transfection, the cells were starved for at least 3 h and then stimulated with different concentrations of UK14,304 (0.01, 0.1 and 1 mM) for 5 min. Stimulation was terminated by addition of 150 ml of ice-cold cell lysis buffer containing 50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 1 Nonidet P-40, 1 mM sodium orthovanadate and protease inhibitor cocktail (Roche). After solubilizing the cells on ice for 20 min, 10 ml of total cell lysates was separated by 12 SDS-PAGE and ERK1/2 activation was determined by measuring the levels of phosphorylation of ERK1/ 2 with phospho-specific ERK1/2 antibodies by immunoblotting [47,48].Intact Cell Ligand BindingCell-surface expression of a2A-AR and a2B-AR in HEK293 cells was measured by ligand binding of intact live cells using the membrane impermeable ligands [3H]-Castanospermine RX821002 as described previously [41,45]. Briefly, HEK293 cells cultured on 6well dishes were transiently transfected with 1 mg of plasmids. After 6 h the cells were split into 24-well dishes pre-coated with poly-L-lysine. Forty-eight h post-transfection, the cells were incubated with DMEM plus [3H]-RX821002 in a total of 300 ml for 60 min at room temperature. In the initial experiments, the cells were incubated with increasing concentrations of [3H]-RX821002 (from 0.3125 to 20 nM) to generate ligand dose-dependent binding curves. Because ligand binding to the receptors is almost saturated at a concentration of 20 nM, this concentration was then used to measure the cellsurface expression 15755315 of the receptors. The binding was terminated and excess radioligand eliminated by washing the cells twice with ice-cold DMEM. The retained radioligand was extracted by digesting the cells in 1 M NaOH for 2 h at room temperature. The liquid phase was collected and suspended inStatistical AnalysisDifferences were evaluated using MedChemExpress Biotin N-hydroxysuccinimide ester one-way ANOVA and posthoc Tukey’s test, and p,0.05 was considered as statistically significant. Data are expressed as the mean 6 S.E.a2-AR Export and Cell-Surface ExpressionResults Differential Inhibition of the Cell-surface Expression of a2A-AR and a2B-AR by Mutation of Leu and a Positively Charged Residue on the ICLThe amino acid sequences of the ICL1 are highly conserved in each a2-AR subty.On coverslips pre-coated with poly-L-lysine in 6-well plates and transfected with 100 ng of GFP-tagged receptors. For co-localization of GFPtagged receptors with the ER marker DsRed2-ER, HEK293 cells grown on coverslips were transfected with 100 ng of GFP-tagged receptors and 100 ng of pDsRed2-ER. The cells were fixed with 4 paraformaldehyde-4 sucrose mixture in PBS for 15 min and the coverslips were mounted with prolong antifade reagent containing DAPI. Images were captured using a Zessis confocal microscope (LSM510) equipped with a 63x objective (NA = 1.3). The colocalization of the receptor with the ER marker DsRed2ER was determined by Pearson’s coefficient 12926553 using the ImageJ JaCoP plug-in as described [46].Cell Culture and Transient TransfectionHEK293 and HeLa cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) with 10 fetal bovine serum (FBS), 100 units/ml penicillin, and 100 units/ml streptomycin and transiently transfected by using Lipofectamine 2000 reagent as described previously [44]. Transfection efficiency was estimated to be greater than 70 based on the GFP fluorescence.Measurement of ERK1/2 ActivationHEK293 cells were cultured in 6-well dishes and transfected with 1 mg of a2A-AR or its mutants as described above. After 24 h transfection, the cells were starved for at least 3 h and then stimulated with different concentrations of UK14,304 (0.01, 0.1 and 1 mM) for 5 min. Stimulation was terminated by addition of 150 ml of ice-cold cell lysis buffer containing 50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 1 Nonidet P-40, 1 mM sodium orthovanadate and protease inhibitor cocktail (Roche). After solubilizing the cells on ice for 20 min, 10 ml of total cell lysates was separated by 12 SDS-PAGE and ERK1/2 activation was determined by measuring the levels of phosphorylation of ERK1/ 2 with phospho-specific ERK1/2 antibodies by immunoblotting [47,48].Intact Cell Ligand BindingCell-surface expression of a2A-AR and a2B-AR in HEK293 cells was measured by ligand binding of intact live cells using the membrane impermeable ligands [3H]-RX821002 as described previously [41,45]. Briefly, HEK293 cells cultured on 6well dishes were transiently transfected with 1 mg of plasmids. After 6 h the cells were split into 24-well dishes pre-coated with poly-L-lysine. Forty-eight h post-transfection, the cells were incubated with DMEM plus [3H]-RX821002 in a total of 300 ml for 60 min at room temperature. In the initial experiments, the cells were incubated with increasing concentrations of [3H]-RX821002 (from 0.3125 to 20 nM) to generate ligand dose-dependent binding curves. Because ligand binding to the receptors is almost saturated at a concentration of 20 nM, this concentration was then used to measure the cellsurface expression 15755315 of the receptors. The binding was terminated and excess radioligand eliminated by washing the cells twice with ice-cold DMEM. The retained radioligand was extracted by digesting the cells in 1 M NaOH for 2 h at room temperature. The liquid phase was collected and suspended inStatistical AnalysisDifferences were evaluated using one-way ANOVA and posthoc Tukey’s test, and p,0.05 was considered as statistically significant. Data are expressed as the mean 6 S.E.a2-AR Export and Cell-Surface ExpressionResults Differential Inhibition of the Cell-surface Expression of a2A-AR and a2B-AR by Mutation of Leu and a Positively Charged Residue on the ICLThe amino acid sequences of the ICL1 are highly conserved in each a2-AR subty.