Figure 4.

Overexpression of PA28γ abrogates glucosamine-mediated proteasomal inhibition and apoptosis. (A) ALVA41 cells were treated with various concentrations of glucosamine for 24 h and Western blot analysis was performed using the indicated antibodies. (B) ALVA41 cells were treated with vehicle or the indicated concentrations of glucosamine for 24 h and Western blot analysis was performed to measure the expression level of PA28γ. The ratios vs. that of control (normalized by γ-tubulin) from the three different experiments were graphed. (C) 24 h after transfection with mock or PA28γ expression vector, ALVA41 cells were treated with vehicle or 1 mM of glucosamine for additional 24 h and PA28γ expression was analyzed using Western blot analysis. A representative image was presented, and the ratios vs. that of control (normalized by γ-tubulin) were noted at the bottom of the image (n = 3). (D) 24 h after transfection with mock or PA28γ expression vector, ALVA41 cells were treated with vehicle or 1 mM of glucosamine for additional 24 h and chymotrypsin-like proteasomal activity was measured. (E) 24 h after transfection with mock or PA28γ expression vector, ALVA41 cells were treated with vehicle or 1 mM of glucosamine for additional 24 h and apoptotic cells were measured using annexin V/PI double staining and subsequent flow cytometry. ^*P < 0.01.