10.1371/journal.pone.0015062.g007 Carla Figueira Bento Carla Figueira Bento Rosa Fernandes Rosa Fernandes José Ramalho José Ramalho Carla Marques Carla Marques Fu Shang Fu Shang Allen Taylor Allen Taylor Paulo Pereira Paulo Pereira Silencing of CHIP stabilized HIF-1α protein in the presence of methylglyoxal in ARPE-19, Cos-7 and RCC4 cells. Public Library of Science 2013 stabilized methylglyoxal cos-7 rcc4 2013-02-20 22:11:00 Figure https://plos.figshare.com/articles/figure/_Silencing_of_CHIP_stabilized_HIF_1_945_protein_in_the_presence_of_methylglyoxal_in_ARPE_19_Cos_7_and_RCC4_cells_/486764 <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>