Figure EV1. Purification of mitochondria from mammalian cells and validation of the PINK1 antibody.

1. Workflow for the preparation and purification of mitochondrial fractions.
2. Quality control for the purity of the extracted mitochondria proteins. Purified mitochondrial fractions (M) and whole cell lysates (WCL) were tested for purity by immunoblotting using the antibodies indicated. For both the purified mitochondria and the whole cell lysate 10% of the total amount of cells was used for the analysis.
3. PINK1 is proteolytically processed by the mitochondrial processing peptidase (MPP) and the presenilin‐associated rhomboid‐like protein (PARL). Domain structure of PINK1 (left panel) and representation of PINK1 species in immunoblots (right panel). MTS: mitochondrial targeting sequence; TMD: transmembrane domain.
4. Validation of the PINK1 antibody in cells silenced for PINK1 expression. HEK 293T cells were transfected with control or PINK1 siRNA. 48 h after transfection, the cells were treated with CCCP (10 μM, 90 min) before harvesting. The cell lysates were analyzed by immunoblotting using the PINK1 antibody D8G3 (rabbit mAb #6946) from Cell Signaling Technology. β‐Actin was used as a loading control.