Figure 6. p135 is a potential substrate of Shp2 phosphatase in the mitochondria.

BaF3 cells were transfected with Shp2 D425A or wildtype Shp2 expressing plasmids through retroviral mediated gene transfer. Transduced cells were sorted based on green fluorescent protein (GFP) that is expressed under a separate promoter contained in the retroviral vector MSCV. Sorted cell pools were treated with H₂O₂ (500 µM) for 20 min. Mitochondria were purified using a kit. Mitochondrial extracts and cytosolic lysates were subjected to anti-Shp2 immunoprecipitation followed by anti-phospho-tyrosine (pY) immunoblotting. Blots were stripped and reprobed with anti-Shp2 antibody.