FIGURE 6.

Effect of down-regulation of Csk on the cannabinoid-inhibited TCR signaling. A, down-regulation of Csk by siRNA. Western blot experiments showing the expression of Csk and actin in IL-4-stimulated Jurkat cells 4 days after siRNA transfection. The cells were either untransfected (−) or transfected with a nonmatching control siRNA (co) or siRNA directed against human Csk (Csk). B, down-regulation of Csk abolishes the inhibition of TCR signaling by cannabinoids. Jurkat cells were transfected with Csk siRNA (above) and control siRNA (below). Four days later, the cells were treated with cannabinoids for 4 h according to the scheme below the gels (THC, 500 nm; MAEA, 500 nm; JWH015, 250 nm). Then the cells received fresh medium and were activated with anti-CD3/anti-CD28 for 2 min as indicated. Representative results monitoring phospho-LAT and actin are shown. C, quantification of Western blot signals of at least two individual experiments performed in duplicate normalized to actin. The samples are compared with the anti-CD3/anti-CD28-activated samples shown in group 2. Secondary comparisons are indicated by brackets. Gray columns, control siRNA; white columns, Csk siRNA. ***, p < 0.001.