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Cells have been fixed in 4% paraformaldehyde for 15min and washed with phosphate-buffered saline (PBS) containing 20mM glycine to quench the aldehyde groups. Immediately after permeabilization with PBS containing 0.05% Triton X-100 for 5min, cells were incubated 1h at space temperature Seliciclib CDK2 with PBS containing 1% bovine serum albumin and had been labelled with the following key antibodies: mouse monoclonal anti-GM130 antibody (1/250, BD Biosciences, Franklin Lakes, NJ, USA), mouse monoclonal antitransferrin receptor antibody (1/200, Invitrogen, Camarillo, CA, USA), or mouse monoclonal anti-LAMP1 antibody (1/200, Enzo, Farmingdale, NY, USA) for 1h, washed, and stained with the secondary Cy3 donkey anti-mouse antibody (1/200, Jackson Immunoresearch Laboratories, Baltimore, PA, USA) plus Hoescht as nuclei marker.
D2LR-YFP was detected by monitoring fluorescence emission at 530nm. The slices had been mounted making use of the mounting references medium and have been observed in the Leica SP2 confocal microscope. Photos have been acquired in a sequential mode using a 63x Plan Apochromat Ketoconazole NA one.four oil aim plus the ideal filter mixture.2.four. ERK 1/2 Phosphorylation AssaysHEK-293T cells, transiently transfected with 1��g of cDNA corresponding to D2LR or D2LR-YFP, were cultured in serum-free medium for 16h just before the addition of 100nM quinpirole for 5min. Cells had been lysed in ice-cold lysis buffer (50mM Tris-HCl pH seven.four, 50mM NaF, 150mM NaCl, 45mM ��-glycerophosphate, 1% Triton X-100, 20��M phenyl-arsine oxide, 0.4mM NaVO4 and protease inhibitor cocktail), and ERK 1/2 phosphorylation was established as indicated elsewhere [11, 12].two.5. cAMP DeterminationHEK-293T cells, transiently transfected with 1��g of cDNA corresponding to D2LR or D2LR-YFP, had been grown in 25cm2 flasks to 80% confluence and incubated in serum-free medium for 16h prior to the experiment.