Fig. 2.

Butyric acid-induced increase in mitochondrial H₂O₂ amounts is correlated to increased heme levels. a Isolated blood mitochondria from 0, 60, and 180 min after butyric acid injection. Qproteome^TM Mitochondria Isolation Kit (Qiagen) was used to isolate blood mitochondria. Pierce® Detergent Removal Spin Columns (Thermo Scientific) was used to purify samples from traces of detergents. Pierce® Microplate BCA Protein Assay Kit-Reducing Agent Compatible Kit (Thermo Scientific) was used to standardize the protein concentration in all samples used. All kits were used according to manufacturer’s recommendation. Anti-MTC02 (Novus Biologicals) is a mitochondria-specific antibody used to verify the purity of the isolated blood mitochondria. Anti-HSP60 (StressMarq Biosciences Inc., Canada) is used to detect cytoplasmic heat-shock protein 60 in the blood mitochondria to further confirm the purity of the mitochondrial samples. Anti-GAPDH (GeneTex) is used to detect the glyceraldehydes-3-phosphate in the blood mitochondria to serve as control. b H₂O₂ amounts in blood mitochondria samples. Red Hydrogen Peroxide Assay Kit (Enzo Life Sciences) was used to measure mitochondrial H₂O₂ amounts according to manufacturer’s recommendation. c Total heme levels in blood mitochondria samples. QuantiChrom^TM Heme Assay Kit was used to measure total mitochondrial heme levels (free heme and heme-proteins) according to manufacturer’s recommendation. In all assays performed, results shown are mean ± SE, n = 5 replicates of six independent samples. Statistical analyses were performed using Student’s t test (^**represents p value < 0.01compared to normal)