Fig 1: Mitochondrial purification from C. burnetii-infected cells.A, schematic representation of mitochondrial purification method from C. burnetii-infected cells. B, continuous sucrose gradient centrifugation of mitochondria isolated from C. burnetiimCherry-infected HeLa cells. A crude mitochondrial sample was taken pre-centrifugation (crude mito). Fractions were collected following ultracentrifugation and analyzed by SDS-PAGE and immunoblotting with the indicated antibodies. Fractions containing the majority of mitochondrial or C. burnetii protein are marked with “mitochondria” or “Coxiella”, respectively. C, mitochondria were isolated from THP-1 cells infected for 3 days with C. burnetiimCherry and purified by continuous sucrose gradient centrifugation and anti-Tom22 separation. Samples were collected after crude mitochondrial isolation (Crude mito, lane 1) and after purification (Post Tom22 purification, lanes 2 and 3). Purified mitochondria were incubated with or without Proteinase K (PK; 50 µg/ml) and analyzed by SDS-PAGE and immunoblotting using the indicated antibodies as the listed organelle markers. Cyt c, cytochrome c; IMM, inner mitochondrial membrane; IMS, intermembrane space; ER, endoplasmic reticulum; OMM, outer mitochondrial membrane; THP-1, human monocyte–derived macrophage cell line.