The cells were fixed with 4% paraformaldehyde?4% sucrose mixture in PBS for 15 min and stained with 4, 6-diamidino-2-phenylindole for 5 min. For colocalization of GFP-tagged receptors with Nutlin-3 the cis-Golgi marker GM130 or with all the plasma membrane marker Na+/K+ ATP-ase, HEK293T cells had been permeabilized with PBS containing 0.2% Triton X-100 for 5 min, and blocked with 5% normal donkey serum for 1 h. The cells have been then incubated with antibodies against GM130 or Na+/K+ ATP-ase at a dilution of 1:one hundred for 1 h. Right after washing with PBS , the cells had been incubated with Alexa Fluor 594-labeled secondary antibody for 1 h at room temperature. The coverslips have been mounted, and fluorescence was detected using a Leica DMRA2 epifluorescent microscope as described previously . Photos have been deconvolved applying SlideBook computer software along with the nearest neighbor deconvolution algorithm . two.7. Co-immunoprecipitation Immuno-precipitation from the receptors was performed in equivalent manner as described . In short, HEK293T cells were cultured on ten cm2 dishes and transfected with three ?g of HAtagged ?2C-AR or ?2B-AR for 48 h. The cells have been washed twice with PBS and harvested. The cells were then lysed with 300 ?l of lysis buffer containing 50 mM Tris?HCl, pH 7.
4, 150 mM NaCl, 1% NP-40, 0.5% sodium deoxycholate, 0.1% SDS and Full Mini protease inhibitor cocktail. Right after gentle rotation for 1 h, samples were centrifuged for 15 min at 14,000 ?g plus the supernatant was incubated with 50 ?l of protein G Sepharose for 1 h at four?C to remove non-specific bound proteins.
plx4720 selleck chemicals Samples have been then incubated with five ?g of anti-GFP antibodies overnight at 4?C with gentle rotation followed by incubation with 50 ?l of protein G sepharose beads for 5 h. Resin was collected by centrifugation and washed 4 instances with 500 ?l of lysis buffer. Immunoprecipitated receptors had been eluted with one hundred ?l of 1xSDS-PAGE loading buffer, separated by 10% SDS-PAGE and visualized by immunoblotting utilizing particular antibodies. two.8. Western Blotting Western blot analysis of protein expression was carried out as previously described . Samples were separated by SDS-PAGE and transferred onto polyvinylidene difluoride membranes. The signal was detected employing ECL Plus in addition to a Fuji Film luminescent image analyzer and quantitated using the Image Gauge system . two.9. Measurement of cAMP production cAMP concentrations have been measured by using cAMP enzymeimmunoassay system as described previously . HEK293T cells on 10 cm2 plates were transfected with 3 ?g ?2C-AR and six hours later had been split into 12-well plates. The cells had been serum straved for 24 hours after which incubated at 37?C or at 30?C in absence or presence of macbecin for the subsequent 18 h. One particular hour prior to stimulation the medium was changed to PBS supplemented with isobutylmethylxanthine . Then the cells had been incubated with ten?8 M UK14304 for five min, followed by stimulation with forskolin for 15 min.