Identification of putative AnxA6 binding partner(s) in rat heart. A. Purified GST-AnxA6 (A6) affinity beads or GST affinity beads (V) were incubated with NP-40 solubilised WHH (1.5 mg) and the bound proteins (interactome) were separated in 12% SDS-PAGE, stained with Coomassie (lane 2) and analysed via LC MS/MS. B. In vitro binding assay: Solubilised whole heart homogenates (WHH) were incubated with GST affinity beads (vector) or AnxA6-GST affinity beads and the bound proteins collected in elute were separated by 10% SDS-PAGE followed by immunoblotting analysis with anti sarcomeric α-actinin antibody. C. In vitro binding assay with lipid depleted WHH: Solubilised WHH or delipidated WHH incubated either with GST-AnxA6 affinity beads or GST (vector) affinity beads and the bound proteins in elute were separated by 10% SDS-PAGE followed by immunoblotting analysis with anti sarcomeric α-actinin antibody. D. Immunoprecipitation: The whole heart homogenate (300 μg) was subjected to immunoprecipitation with anti sarcomeric α-actinin antibody followed by western blotting with anti-AnxA6 antibody.
Mishra et al. BMC Cell Biology 2011 12:7 doi:10.1186/1471-2121-12-7