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Human primary CD4+ T cells were pre-incubated for 15 minutes at 37°C with 30 ?M ML-7, the MLCK inhibitor, or the vehicle (DMSO) alone. Cells were then activated for 10 to 30 minutes with anti-CD3/anti-CD28 beads in the presence of ML-7. Phosphorylation of the MLC was measured by Western blot analysis. The gp96 protein in each lane is shown as a loading control, Compared to control (DMSO), the cells incubated with ML-7 show less phosphorylation of MLC demonstrating that ML-7 is inhibiting MLCK in our conditions ,