Figure 2. Proteasome limits IL-6 gene transcription mediated by NF-κB subunit, p65.

(A). ILU-18 cells were either pretreated with SN50 (18μM) for 1 hour or left untreated. At the end of 1 hour, these cells were either exposed to PV (100μM) or left untreated for 18 hours. At the end of 18 hours, cell lysates were obtained and luciferase activity was determined employing the Promega luciferase assay kit. For panels (A, C, D and E), fold induction represents a ratio of luciferase activity obtained in treatment-induced to that of untreated cells. Data represents mean ± standard deviation from four independent experiments, following normalization. Statistically significant differences are denoted by **.

(B). Murine Embryonic Fibroblasts (p50^−/− and p65^−/−), either untreated (UN) or treated with PV (100μM) for 4 hours with or without pretreatment with Acla (0.25μM) for 2 hours were used to isolate total RNA. Equal amounts of RNA were analyzed by RT-PCR. β-actin was used as a loading control.

(C). ILU-18 cells were either pretreated with Aclacinomycin (0.25μM) or left untreated. At the end of 2 hours, cells were stimulated with PV (100μM) for 18 hours or left untreated. Lysates obtained were evaluated for luciferase activity.

(D). ILU-18 cells were pretreated with either MG132 (1μM) or PSI-1 (20μM) for 2 hours or left untreated. At the end of 2h, cells were activated with PV (100μM) for 18 hours. Cells treated with MG132 or PSI-1 for 20 hours served as controls. Lysates obtained were evaluated for luciferase activity.

(E). ILU-18 cells were either pretreated with 0.25μM Acla or left untreated. At the end of 2 hours, cells were subjected to hypoxia (H). Cells underwent reoxygenation (R) for 18 hours, either in the presence or absence of 0.25μM Acla. This resulted in the following samples: H/R, Acla+H/R, and H+Acla/R. Lysates obtained were evaluated for luciferase activity.