Res Commun Mol Pathol Pharmacol 1997;96:71C87 [PubMed] [Google Scholar] 36. additional 2 h. Immunoprecipitates were washed five times with TBS, and the residue TBS buffer was removed. The activity of purified PTP1B was assayed with a PTP1B assay kit (Calbiochem) according to the manufacturers protocol. Briefly, the phosphopeptide substrate IR5 (containing a sequence from the insulin receptor subunit domain that must be autophosphorylated to achieve full receptor kinase activation) was added to a final concentration of 75 mol/L in a total reaction volume of 100 L in the assay buffer. The sample mixtures were incubated for 30 min at 30C. After the reaction, 60-L aliquots were placed into half-area 96-well plates, and 25 L red reagent plus 40 L Osthole assay buffer were added to each sample well and gently mixed. After incubation at room temperature for 30 min, the absorbance was measured at 620 nm with a plate reader. Imaging. The immunocytochemical labeling was examined with a confocal microscope as described previously (4,12C15). Confocal imaging was performed with a Leica SP5 X imaging system equipped with ultraviolet (405 nm), tunable (470C670 nm) white light and argon ion lasers (458, 477, 488, 496, 514 nm); 40 and 60 1.4 numerical aperture oil-immersion lenses were used to acquire optical sections. During image acquisition, the individual microscopic field was selected to include a similar number of cells but was otherwise random. To quantify fluorescence intensity, the images from randomly selected microscopic fields containing a similar number of nuclei staining were outlined, and CD244 the integrated fluorescence intensities were measured with Image J software. In the case (Fig. 5< 0.001 compared with the remaining groups. < 0.05 compared with either vehicle or control group. Results were the sum of three independent experiments, with triplicates for each experiment. Statistical analysis. Data are presented as mean SEM. Statistical comparisons among different groups were made with one-way ANOVA with Student-Newman-Keuls post hoc testing. Statistical significance is defined as 0.05. RESULTS NO production regulates FITC-insulin uptake. We first examined the effect of l-shows that compared with control, pretreating bAECs with l-NAME strongly Osthole inhibited FITC-insulin uptake (< 0.05). Conversely, pretreatment of bAECs with 500 mol/L l-arginine (the substrate of eNOS) for 30 min significantly increased FITC-insulin uptake (Fig. 1and and < 0.05) (Fig. 1and and and < 0.05 compared with EBM + FITC-insulin but > 0.05 compared with EBM (incubated in the basal medium without FITC-insulin). < 0.001 compared with all remaining groups. < 0.05 compared with EBM group, < 0.01 compared with SNP group, and < 0.001 compared with L-ARG group, but > 0.05 compared with D-ARG and L-ARG + LNA groups; **< 0.001 compared with all remaining groups. < 0.01 compared with remaining groups. Open in a separate window Open in a separate window Open in a separate window FIG. 6. Effects of knockdown of Txnip on insulin uptake. bAECs were transfected with either Txnip siRNA or scrambled control siRNA. Forty-eight hours after the transfection, cells were processed for Western blotting or serum starved for 6 h followed by incubation with or without 50 nmol/L FITC-insulin 0.3 mol/L SNP for 30 min before they were fixed and doubly stained with anti-FITC (red, revealed by Cy3) and anti-Txnip (green, revealed by Cy2) primary antibodies. < 0.01 compared with scrambled Osthole control. and < 0.05 compared with remaining groups; #> 0.05 compared EBM + FITC-insulin group (FITC-insulin treated without transfection of siRNA). and < 0.05 compared with remaining groups. = 3); no statistical difference was found between treatments. CtsiRNA, control siRNA; INS, insulin; TxsiRNA, Txnip siRNA. Next, we examined Osthole the effect of SNP on 125I-insulin TET with a Transwell device (4,14). Figure 2 shows that compared with control, adding SNP increased 125I-insulin TET by 40% at both 10 and 60 min (< 0.05 for each time point). In aggregate, these data suggest that the NO donor SNP may directly promote insulin transport in an eNOS activity-independent fashion. Open in a separate window FIG. 2. SNP promotes insulin TET. 125I-insulin Osthole 200 pmol/L alone or in the presence of either 0.3 mol/L SNP or vehicle was added into the top chamber of Transwell plates, and samples were removed from the bottom chamber at both 10 and 60 min for measurement of the amount of 125I-insulin transported. Percent transport of total added.