2003), but would not affect the mobility on Western blots. determined using the Coomassie dye binding method (Bradford 1976). Protein fractions were taken up in Laemmlis sample buffer with 1?mM dithiotheitol and heated at 60C for 10?min, and equal amounts of protein (45?g) were loaded per lane on SDS-6% polyacrylamide gels for electrophoresis (50?V for 30?min and then 120?V for 80?min). The proteins were transferred onto polyvinylidine difluoride membranes in Towbins buffer (24?mM Tris Base, 192?mM glycine, 20% methanol, pH?8.3) for 1?h in the cold at 95?V using a Bio-Rad Trans-Blot Cell (BioRad Laboratories) with an ice pack. Blots were rinsed three times (5?min each) with Tris-buffered saline (TBS; 20?mM Cyanidin chloride TrisCCl, pH?7.4, 137?mM NaCl) and incubated with blocking solution (5% nonfat dry milk, 5% goat serum in TBS) at room temperature for 1?h. Rabbit polyclonal to MCAM Blots were then incubated for 1?h at room temperature with an antibody (1:1,000) raised against a peptide comprising amino acids 1422C1433 of human nNOS (Bredt et al. 1991; Nakane et al. 1993) or amino acids 1418C1429 of mouse nNOS (Ogura et Cyanidin chloride al. 1993). The blots were washed three times with TBS-T (TBS containing 0.1% Tween 20), incubated with horseradish peroxidase-coupled anti-rabbit IgG (1:8,000) for 1?h at room temperature, and washed five times with TBS-T followed by five times with NANOpure water. Immunoreactive bands were detected by enhanced chemiluminescence and exposure to Hyperfilm at various Cyanidin chloride time intervals to obtain optimal signals. The blots were developed using a Kodak M35A X-OMAT processor (Rochester, NY, USA). Band densities were quantified using an Alpha Innotech Imager with AlphaEase software (Alpha Innotech, San Leandro, CA, USA). The average number of pixels per enclosed area after background correction was normalized to the control samples as 100%. The data were tested for statistically significant differences using one-way ANOVA and Dunnetts post hoc test or Students test (Prism version 4.00, GraphPad Software, San Diego, CA, USA). Calcium imaging N18TG2 cells were cultured on 25-mm glass cover slips in six-well plates for 48?h until 85% confluent. The cover slips were mounted in an Attofluor Cell Chamber (catalog no. A-7816, Molecular Probes). Cells were loaded with 5?M Fluo-4?AM at room temperature, and the cover slips were washed three times with PSS before exposure to agonists. One milliliter of PSS was maintained in the chamber throughout the experimental period by removing 100?l of PSS before each addition of 100?l of cannabinoid agonists (0.3 nMC1?M, final concentration). Serially increasing concentrations of agonists were added to the chamber every 60? s over a time course of 360?s. Intracellular Ca2+ measurements were taken from images containing up to 40 cells and captured at a rate of one frame per 983?ms, using a Zeiss LSM 510 Confocal microscope with LSM 510 software (Zeiss, Thornwood, NY, USA). Excitation and emitting wavelengths were 488 and 514?nm, respectively. The baseline was established as the fluorescence at the initial 30?s prior to adding drugs. For every experiment, the effects of cannabinoid agonists were compared to the dose-dependent response to bradykinin. The data were analyzed, and graphs were prepared using Prism 4.00. Results CB1 agonists stimulate NO production in N18TG2 cells N18TG2 neuroblastoma cells loaded with DAF-FM-diacetate were treated with cannabinoid receptor agonists CP55940, WIN55212-2, and the metabolically stable anandamide analog MetAEA (Fig.?1a). The low background fluorescence indicates that the cellular production of NO does not occur constitutively in these cells. Over the 20-min period of Cyanidin chloride NO accumulation, all three cannabinoid receptor agonists produced maximal NO-DAF-FM fluorescence at 10 nM concentrations, indicating that the cells were extremely sensitive to agonist stimulation (Fig.?1b). Pretreatment with the CB1 antagonist rimonabant reduced the NO-DAF-FM fluorescence in response to 10 nM CP55940 or WIN55212-2 and 1?M MetAEA to the unstimulated control levels (Fig.?1c), indicating that the Zero production could possibly be related to CB1 receptor stimulation. Prior studies had showed which the CB2 receptor isn’t portrayed in N18TG2 cells (Jones et al..