Fig. 6

AGR2 regulates H6PD protein expression and enhances its enzyme activity. A–C HEK-293 cells were transfected with plasmid for H6PD-MYC in the absence or presence of plasmid for AGR2-FLAG, followed by determination of protein expression by immunoblotting and measurement of H6PD enzyme activity. A) Relative H6PD-MYC protein levels in microsomes of HEK-293 cells normalized to the β-Actin loading control, determined by densitometry analysis. H6PD-MYC expression was to β-Actin protein levels, relative to that of microsomes of H6PD-MYC transfected cells. B Representative western blot analysis of H6PD-MYC and AGR2-FLAG protein levels in microsomes of HEK-293 cells transfected with H6PD-MYC alone or together with AGR2-FLAG. Membranes were probed with anti-MYC antibody for H6PD and anti-FLAG antibody for AGR2 detection. C H6PD activity in microsomes of HEK-293 cells transfected with H6PD-MYC alone or together with AGR2-FLAG. In each independent experiment, the results were normalized to the activity of microsomes of HEK-293 cells transfected with H6PD-MYC. D-H) MCF7 cells were treated for 72 h with control siRNA (siCTRL), siRNA against AGR2 (siAGR2) or H6PD (siH6PD), followed by immunoblotting analysis and determination of H6PD activity in microsomal preparations. D AGR2 protein levels were normalized to the β-Actin loading control and are relative to siCTRL, determined by densitometry analysis. E) Representative western blot of (D). F H6PD protein levels normalized to β-Actin loading control and relative to siCTRL. G Representative western blot of (F). H H6PD activity in microsomes of MCF7 cells transfected with siCTRL or siAGR2. In each independent experiment, results were normalized to H6PD activity in microsomes of siCTRL-transfected MCF7 cells. A, C, D, F, H Data represent mean ± SD from three independent experiments. Mann–Whitney U test was performed, p-value: * = < 0.05