Figure 3

NDRG2 downregulates GLUT1 by promoting its ubiquitination. (A), (C) and (E) SK-BR-3 cells were infected with an adenovirus carrying N-myc downstream-regulated gene 2 (Ad-NDRG2) at 1, 5 and 10 multiplicity of infection (MOI) or Ad-LacZ for 48 hours. (B), (D) and (F) T-47D cells were transfected with NDRG2 small interfering RNA (siRNA) 10, 25 and 100 pmol or control siRNA for 48 hours. Next, cell proteins or mRNA were extracted and analysed by immunoblotting (A) and (B) or by real-time PCR (C) to (F). β-actin was used as a loading control. (C) – (F) The data presented are the means ± SD of three independent experiments; error bars represent SD from 3 replicative wells. *P < 0.05 and **P < 0.01 versus control group. (G) SK-BR-3 cells were infected with 10 MOI Ad-NDRG2 or Ad-LacZ for 48 hours and then treated with 2 μM, 6 μM or 8 μM MG-132 for 4 hours. Next, the protein was extracted and analysed by immunoblotting. (H) Cell fractions were prepared from the SK-BR-3 cells infected with 10 MOI Ad-NDRG2 or Ad-LacZ for 48 hours, and the membrane and cytosolic fractions of endogenous glucose transporter 1 (GLUT1) protein were detected. Tubulin and β-actin served as loading controls. (I) SK-BR-3 cells were transfected with hemagglutinin (HA)-ubiquitin plasmid for 6 hours and infected with Ad-NDRG2 or Ad-LacZ for another 48 hours. Subsequently, the cell lysates were collected and analysed by immunoprecipitation (IP) and immunoblotting with GLUT1 and HA antibodies. WB, Western blot.