Hypoxia increases c-Aco activity but does not regulate m-Aco protein accumulation or activity. (A-C) HEK293 cells were grown for the indicated times under hypoxic conditions or under normoxia with Df (200 μM for 16 hours) or FAC (150 μg/mL for 5 hours). (A) Bar graph shows the mean aconitase activity of mitochondrial and cytosolic cell fractions ± SEM, n = 6 (0, 1, 7, and 16 hours hypoxia) or n = 4 (FAC and Df). The ^* indicates points that differ from the control group (0 hour hypoxia) with P < .007 using a Student t test. Aconitase activity was assayed by measuring the change in absorbance at 240 nm over time as cis-aconitate is converted to isocitrate.⁴²  (B) Immunoblot analysis with chicken anti-IRP1³⁶  or rabbit anti-m-Aco antibodies to determine the purity of the mitochondrial and cytosolic fractions. Western analysis was performed for all experiments with a representative blot of the 16-hour hypoxia time point shown. Cyto indicates cytosolic fraction; M, mitochondrial fraction. (C) Immunoblot analysis of cytosolic cell extracts with rabbit anti-m-Aco antibodies. Experiment was performed 3 times with a representative blot shown.