Extended Data Fig. 3. A fraction of most members of the small heat shock protein family resides in the mitochondrial intermembrane space.
Proteinase K protection assay on mitochondria isolated from non-heat shocked cells: HEK293T (human embryonic kidney cells), NSC-34 (motor neuron-like cells), Neuro-2A (neuronal crest-derived), SH-SY5Y (neuroblastoma cells), COS1 (African green monkey derived kidney cells), A498 (renal carcinoma cells), and healthy volunteer-derived human lymphoblasts and fibroblast, or isolated from the heart of a C57BL/6 mouse. Isolated mitochondria were resuspended in a hypotonic (osmotic swelling) buffer to generate mitoplasts. Proteinase K (10 μg/ml) was used to digest accessible proteins. Samples were analyzed by SDS-PAGE followed by immunoblotting using anti-HSPB antibodies and anti-TOM20 (OM), anti-AIFM1 (IMS), anti-HCCS (IM), and anti-SDHA (matrix) antibodies. Results are representative of two replicates. Unprocessed blots are available in source data.