Silencing of CHIP stabilized HIF-1α protein in the presence of methylglyoxal in ARPE-19, Cos-7 and RCC4 cells.

<p>(A) ARPE-19 cells were infected with pAd shRNA-hCHIP for 48 hours. Cells were subsequently lysed and protein extracts were immunoblotted against endogenous CHIP and actin. (B) ARPE-19 cells were co-infected with pAd V5-hCHIP and pAd shRNA-hCHIP for 24 hours. The cell lysates were analyzed by western blotting using anti-V5 and anti-actin monoclonal antibodies. (C) ARPE-19 cells were infected with pAd shRNA-hCHIP for 48 hours and during the last 6 hours of incubation, cells were subjected to hypoxia in the presence of MGO (3 mM for the last 70 minutes). Cell lysates were used to immunoprecipitate HIF-1α and the immunoprecipitates were probed with antibodies against HIF-1α and ubiquitin (P4D1). The data in the graph represents the mean ± SD of at least three independent experiments. ** p<0.01, significantly different from control (one-way ANOVA with the Dunnet's comparison test). (D) RCC4 VHL<sup>-/-</sup> cells, infected with pAd shRNA-hCHIP for 48 hours, were treated with MGO (3 mM for 70 minutes). Cell lysates were used to immunoprecipitate HIF-1α and the immunoprecipitates were blotted against HIF-1α and ubiquitin (P4D1). (E) Cos-7 cells were infected with pAd shRNA-hCHIP for 48 hours and, during the last 6 hours of incubation, cells were subjected to hypoxia and treated with MGO (3 mM for the last 70 minutes). Cell lysates were used to immunoprecipitate HIF-1α and the immunoprecipitates were probed for HIF-1α and ubiquitin (P4D1). IP controls were carried out both with no antibody and with an irrelevant mouse IgG1 antibody (for example anti-GFP). (F) ARPE-19 cells were grown in 15 mM (basal DMEM: F12 medium) or 40 mM of D-glucose during 10 days. After 8 days of incubation, cells were infected with pAd shRNA-hCHIP for 48 hours. During the last 6 hours of incubation, cells were subjected to hypoxia and subsequently lysed. Proteins were blotted against HIF-1α and actin. The word “Mock” in the figures refers to a control with a scrambled shRNA sequence.</p>