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>