Methamphetamine induction of IL-6 and IL-8 is mediated via NF-κB pathway. (A) The proteins from the nucleus and cytoplasm of SVGA cells were collected after various time-periods as indicated and the translocation of p50 was measured by comparing the expression in the nucleus and cytoplasm. The expression in the nucleus and cytoplasm were normalized with laminB and β-Tubulin, respectively, as housekeeping genes. (B) The bar chart represents the mean ± SE of three independent experiments and the blot is a representation of three experiments. (C) SVGA cells were treated with 500 μM of MA for the specified time and the total cell lysates were collected as mentioned in Materials and Methods. Total IκB-α was used as an experimental control and actin was used as a loading control. The blot is representative of three independent experiments. SVGA astrocytes were treated with 10 μM of SC514 for one hour prior to MA treatment every day for three days and the total mRNA was isolated 24 hours after the last dose. The expression levels of IL-6 and IL-8 were measured with real time RT-PCR and the percent mRNA expression of IL-6 (D) and IL-8 (E) were calculated relative to MA-mediated expression levels. SC514-treated cells did not alter the basal expression levels of IL-6 and IL-8 (data not shown). Each bar represents the mean ± SE of three experiments with each experiment performed in triplicate. The statistical significance was calculated using student's t-test and * and ** denotes P ≤ 0.05 and ≤ 0.01, respectively. MA, methamphetamine.