Figure_6.tif (830.37 kB)

CHIP polyubiquitinated HIF-1α in the presence of methylglyoxal.

figure

posted on 2013-02-20, 22:10 authored by Carla Figueira Bento, Rosa Fernandes, José Ramalho, Carla Marques, Fu Shang, Allen Taylor, Paulo Pereira

(A and B) ARPE-19 cells were transiently transfected with the CHIP wt c-myc plasmid and treated with MG132 (20 µM) for 4 hours in the absence or presence of MGO (3 mM for 40 or 70 minutes). HIF-1α (A) or c-myc (B) were immunoprecipitated and the immunoprecipitates were probed for c-myc and HIF-1α. (C) ARPE-19 cells were transfected with CHIP wt c-myc or with the dominant negatives CHIP K30A c-myc and CHIP H260Q c-myc, simultaneously or separately, and treated with MG132 (20 µM) for 41 hours in the presence or absence of MGO (3 mM for 70 minutes). HIF-1α was then immunoprecipitated and the immunoprecipitates were blotted against HIF-1α, c-myc and ubiquitin (P4D1). (D) ARPE-19 cells were transfected with CHIP wt c-myc or with the dominant negative mutants CHIP K30A c-myc and CHIP H260Q c-myc, simultaneously or separately, and subjected to hypoxia for 6 hours in the presence or absence of MGO (3 mM for 70 minutes). HIF-1α was then immunoprecipitated and the immunoprecipitates were blotted for HIF-1α and ubiquitin (P4D1). (E) ARPE-19 cells were transfected with CHIP wt c-myc, CHIP K30A c-myc or/and CHIP H260Q c-myc dominant negatives, either simultaneously or separately, and subjected to hypoxia for 6 hours in the presence or absence of MGO (3 mM for 70 minutes). Proteins were separated by SDS-PAGE, transferred to PVDF membranes and probed for HIF-1α and actin. (F) ARPE-19 cells were treated with MG132 (20 µM for 4 hours) either in the presence or absence of MGO (3 mM for 40 or 70 minutes). HIF-1α was immunoprecipitated and the immunoprecipitates were probed using antibodies against HIF-1α, Hsp70 and Hsp40. (G) ARPE-19 cells were transfected simultaneously with CHIP-c-myc and V5-tagged Hsp40 and/or HA-tagged Hsp70. Cells were subsequently treated with MG132 (20 µM) for 4 hours and MGO (3 mM) for the last 70 minutes of incubation. HIF-1α was immunoprecipitated and the immunoprecipitates were blotted against HA, V5 and c-myc. IP controls were carried out both with no antibody and with an irrelevant mouse IgG1 antibody (for example anti-GFP). The word “Mock” in the figures refers to a control with an empty vector.