Figure 6.

Effects of minocycline on activation and expression of ASK1 in vitro. (A) ASK1 was activated after three hours of SNP treatment, as shown by an increase in phosphorylation of MBP in in vitro kinase assay. With minocycline pretreatment, decreased phosphorylation of MBP by immunoprecipitated ASK1 was observed (*p = 0.033 for 5 μg/mL, all higher doses also resulted in significant decreases; one sample t-test). A corresponding dose-dependent decrease in the amount of ASK1 immunoprecipitated from minocycline-treated cells was quantitated in metabolic labeling experiments (*p = 0.035 for 5 μg/mL, all higher doses also resulted in significant decreases; one sample t-test) (B) Western blot analysis for ASK1 illustrates a dose-dependent decrease in total ASK1 present in whole cell lysates, normalized to actin expression (*p = 0.050 for 5 μg/mL, all higher doses also resulted in significant decreases; one-sample t-test). (C) RT-PCR for ASK1 normalized to GAPDH showed no decrease in levels of ASK1 transcript produced with minocycline treatment (p > 0.05 for all comparisons; one sample t-test). All results are representative of three or four independent experiments, with error bars representing the SEM. Values shown are percent activation or expression relative to activated samples (SNP treated, no minocycline).