Extended Data Fig. 5: BAG3 is recruited to mitochondria upon heat shock but is not imported into mitochondria.
From: Small heat shock proteins operate as molecular chaperones in the mitochondrial intermembrane space
a, HeLa cells were subjected to heat shock (42 °C) for 1 h. Cells were collected afterward and the mitochondrial fraction was separated from the cytosolic fraction. Using immunoblotting we verified the abundance of BAG3 and sHSPs in each fraction. Tubulin was used as a marker for the cytosol and VDAC1 was used as a marker for mitochondria. The NP-40-soluble whole cell lysate (WCL) was used as a reference. b, Proteinase K protection assay on mitochondria isolated from HeLa cells. 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-BAG3, anti-TOM20 (OM), anti-AIFM1 (IMS), anti-HCCS (IM), and anti-SDHA (matrix) antibodies. c, BAG3 knockout HeLa cells were rescued with wild type or delta-IPV BAG3-GFP (deficient in binding to HSPB8) and subjected to proteinase K protection assay as in panel B. Samples were analyzed by SDS-PAGE followed by immunoblotting using anti-BAG3, anti-TOM20 (OM), anti-AIFM1 (IMS), anti-HCCS (IM), and anti-HSP60 (matrix) antibodies. Results are representative of two (a,b) or three (c) replicates. Unprocessed blots are available in source data.
