Journal: eLife

Article Title: Augmenter of liver regeneration regulates cellular iron homeostasis by modulating mitochondrial transport of ATP-binding cassette B8

doi: 10.7554/eLife.65158

Figure Lengend Snippet: ( A ) mRNA (n = 6) and ( B ) protein (n = 3) levels of Alr in wild type (WT) mouse embryonic fibroblasts (MEFs) with or without Alr downregulation. GPAT protein levels (n = 3, C ), xanthine oxidase (XO, n = 6, D ), and cytosolic aconitase (n = 6, E ) activities in cells treated with Alr siRNA. Mitochondrial complex I (n = 7–8, F ) and complex II activity (n = 7–8, G ) in WT MEFs with Alr downregulation. Cells were harvested 48 hr after transfection for RNA analysis and 72 hr after transfection for protein analysis. Quantification of western blotting images is shown in the same panel. Data are presented as mean ± standard error mean (SEM). *p<0.05 by ANOVA. N.S. = not significant.

Article Snippet: The aconitase activity in the concentrated fraction was measured using Aconitase Activity Assay kit (MitoScience).

Techniques: Activity Assay, Transfection, Western Blot

Journal: eLife

Article Title: Augmenter of liver regeneration regulates cellular iron homeostasis by modulating mitochondrial transport of ATP-binding cassette B8

doi: 10.7554/eLife.65158

Figure Lengend Snippet: Tfrc mRNA levels (n = 6, A ) and transferrin-dependent iron uptake (n = 4–6, B ) were measured in wild type (WT) mouse embryonic fibroblasts (MEFs) with endogenous Alr downregulation and concurrent overexpression of different ALR isoforms. Cytosolic aconitase (n = 7–8, C ) and XO activities (n = 5–6, D ), and GPAT protein levels (n = 3–6, E ) are measured in cells with downregulation of endogenous ALR and overexpression of various ALR isoforms. # indicates the band corresponds to GPAT protein. ( F ) Quantification of panel E . EV = empty vector. ALR short = cytosolic isoform of ALR lacking the first 80 amino acids. Data are presented as mean ± SEM. Cells were infected with lentivirus overexpressing the respective ALR isoforms, and transfected with 3’UTR-specific ALR siRNA 24 hr after lentivirus infection. mRNA levels were assayed 48 hr after siRNA transfection, and protein levels and cytosolic XO activity were assayed 72 hr after siRNA transfection. *p<0.05 by ANOVA with post hoc Tukey comparison. N.S. = not significant.

Article Snippet: The aconitase activity in the concentrated fraction was measured using Aconitase Activity Assay kit (MitoScience).

Techniques: Over Expression, Plasmid Preparation, Infection, Transfection, Activity Assay, Comparison