Extended Data Fig. 4. Overexpression of untagged small heat shock proteins demonstrates that a fraction of most family members reside in the mitochondrial intermembrane space.
Proteinase K protection assay on mitochondria isolated from non-heat shocked HeLa cells that were transiently transfected with untagged small heat shock proteins. This allowed us to assess the mitochondrial localization of tissue-specific sHSPs like HSPB4 (expressed in the eye lens) and HSPB9 (expressed in the testis). Isolated mitochondria were resuspended in a hypotonic (osmotic swelling) buffer to generate mitoplasts. Proteinase K (10 μg/ml) was added 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.