Figure 4
S1P 5 knockdown and downstream effects on endothelial S1P receptor expression. (a) A lentiviral shRNA technique was used to knockdown the S1P5. Treatment of the brain ECs with specific shRNA clone E2 resulted in a non-detectable level of S1P5 RNA as measured by qPCR. S1P5 RNA expression levels in control cells were set at 100%. (b) Western blot analysis shows a 43.1% ± 2.12% reduction in S1P5 protein levels when compared to control cells. (c) S1P receptor characterization in S1P5 knockdown cells show reduction in RNA expression levels of all S1P receptors where S1P1 is reduced by 61% (non-targeting control:100.0% ± 8.5, S1P5 knockdown: 38.9% ± 1.8), S1P2 by 39% (non-targeting control: 100.0% ± 0.6, S1P5 knockdown: 60.8% ± 3.3), and S1P3 by 87% (non-targeting control: 100.0% ± 5.3; S1P5 knockdown 13.3% ± 0.8). Again, S1P4 remains undetectable (n.d.) Values represent the mean ± SEM of n = 3. Statistical significance (t-test) is indicated with asterisks: **P < 0.002 and ***P < 0.001.