Figure 1.

Proteasome inhibitors stimulate COX-2 protein expression via enhancing gene transcription in NCI-H292, A549, and AGS epithelial cells. (A) Cells were treated with various concentrations of MG132, PSI-1, or lactacystin for 16 h, and then whole-cell lysates were prepared and subjected to Western blotting using antibodies specific for COX-2 or actin as described in Materials and Methods. (B) NCI-H292 cells were treated with 25 μM MG132 for the indicated time, and then whole-cell lysates were prepared and subjected to Western blotting using antibodies specific for COX-2 or actin, or mRNA was harvested and RT-PCR was performed to quantify COX-2 mRNA. Fold of induction of COX-2 protein or mRNA expression normalized with actin or β-actin was quantified using ImageQuant. (C and D) Cells were treated with MG132, PSI-1, or lactacystin for 16 h. Whole-cell lysates were immunoprecipitated with anti-COX-2 or anti-ubiquitin antibody, and then the precipitates were subjected to SDS-PAGE and Western blotting using anti-COX-2 (C) or anti-ubiquitin (D) antibody. Results are representative of three independent experiments.