Figure 4. Ibudilast inhibits Tat-induced TNFα production in a protein phosphatase dependent manner.
A, BV-2 cells (1.2×105) were left untreated (NT) or were treated with Tat (100 nM) for 8 h with or without pre-treatment for 30 min. with ibudilast (Ib, 50 µM) or vehicle (Veh) alone or together with the protein phosphatase inhibitor, okadaic acid (Oka, 50 nM). TNFα release was measured by ELISA. The Tat+Veh-treated samples were set to 100% and all other samples were compared to this value (the TNFα concentration for this sample was 1861 pg/mL). Results are shown as mean ± SEM of values derived from three replicates from a single representative experiment; two total experiments were performed. Statistical significance is indicated, as compared to Tat+Veh-treated cells (p<0.001, ***) or as compared to Tat+Ib-treated cells (p<0.01, ##). B, Serine/Threonine phosphatase activity was measured in whole cell lysates from BV-2 cells (4.6×105) treated with ibudilast (50 µM) for the indicated periods of time. Results are shown as mean ± SEM of values derived from three replicates from a single representative experiment; two total experiments were performed. Statistical significance (p<0.001) is indicated, ***. C, BV-2 cells (1.2×105) were left untreated (NT) or were treated with ibudilast (50 µM) for 8 h or 24 h, as indicated. Whole cell lysates were subjected to immunoblot analysis using either PP2Ac-specific (upper panel) or α-Tubulin-specific (lower panel) antibodies. The results of a single representative experiment are shown; two total experiments were performed.