FIGURE 5.

Effects of VPAT gene knockdown on endogenous expression of VPAT, polyamine storage, and release from BMMCs. For the VPAT gene knockdown experiment, BMMCs were treated with VPAT-specific siRNA (RNAi) or AllStars negative control siRNA (control) as described under “Experimental Procedures.” a, quantitative PCR analysis of VPAT (top) and VMAT2 (bottom) mRNA expression. Values are shown relative to G3PDH. Data are means ± S.E. (error bars); n = 3 technical replicates. Essentially similar data were obtained from biological replicates (data not shown). ***, p < 0.001. b, immunohistochemical detection of VPAT and VMAT2 in control or RNAi-treated BMMCs. Bars, 10 μm. c, ATP-dependent spermidine uptake by membrane fraction derived from RNAi-treated BMMCs. Spermidine uptake is shown relative to that in the absence of ATP (0.20 nmol/mg protein) as 100%. d, polyamine release from BMMCs stimulated with IgE/antigen (left and middle). Histamine release from BMMCs stimulated with IgE/antigen in the presence or absence of 1 mm spermine (right). Data are means S.E.; n = 3–4 technical replicates. Essentially similar data were obtained from biological replicates (data not shown). *, p < 0.05; **, p < 0.01; ***, p < 0.001.