Calbiochem proteoextract subcellular proteome extraction kit

For isolation of the cytoskeletal fraction, the Calbiochem ProteoExtract Subcellular Proteome Extraction Kit was used (EMD Millipore, Billerica, Massachusetts, USA), following a previously described detergent-based protocol (Boateng et al. 2007 (link)). The remaining myofibrillar cytsokeleton was immunostained with a-actinin antibody (1:200, Cell Signaling Technology, Inc., Danvers, Massachusetts, USA), and either with a p-FAK (Tyr397) antibody (1:200, Cell Signaling Technology, Inc., Danvers, Massachusetts, USA) or a PIP2 antibody (1:200, mouse IgG, Abcam, Cambridge, Massachusetts, USA).
Cardiomyocytes were observed by microscopy (Observer Z1, Zeiss), and by confocal microscopy (LSM 710; Zeiss). Cell surface areas were measured by ImageJ software. In each case, 3 independent experiments were performed, values were calculated, and 20 cells from each condition were randomly chosen and used to calculate cell areas. Experiments were repeated at least 3 times on PDMS (100 kPa, 400 kPa) or glass. The selective extraction method for the subcellular components on the polyacrylamide hydrogels detached the cells from the surface. Efforts to retain cell attachment by shortening the exposure times and altering the detergents were unsuccessful.

For subcellular fractionation of myocytes, the Calbiochem ProteoExtract Subcellular Proteome Extraction Kit was used (Catalog No. 539790; EMD Millipore, Billerica, MA), following a previously described detergent-based protocol [32 (link)]. Cellular proteins were sequentially extracted into four compartments: cytosolic, membrane/organelles, nuclei, and cytoskeleton. Digitonin-EDTA was used to remove the cytosol. Triton-EDTA was used to remove the membrane-organelle fraction. Tween/deoxycholate/benzonase was used to remove the nuclei. Finally, SDS was used to remove the cytoskeleton. Cells were briefly washed three times in PBS between each extraction fraction to prevent cross-contamination. After each fraction, cells were observed by light microscopy to ensure that they were still attached to the dish. The accuracy of the fractionation method was verified with antibodies to well-documented subcellular distribution markers [heat shock protein (Hsp)70 for cytosol, β1-integrin for membrane, H2B for nucleus, and tropomyosin for myofibrils].