Flp In HEK293 stable cell lines expressing either Flag alone, PINK1-Flag Wildtype (WT), PINK1-Flag Kinase dead (KD) or phospho dead mutant PINK1- flag T257A, were induced for expression with doxycyclin for 24 hrs. The cells were stimulated with either DMSO or CCCP for 3hrs and subjected to mitochondrial fractionation. 0.5mg of mitochondrial extracts were immunoprecipitated with 5ul of anti-flag agarose beads to immunoprecipitate recombinant PINK1. Proteins were resolved by 8% SDS-PAGE and subjected to western blot analysis. Proteins were probed with S114D phospho T257 antibody (3rd bleed). The antibody recognises mitochondrial Wild-type PINK1 only upon CCCP stimulation. It does not recognise PINK1 KD. Moreover, the specificity of the antibody is confirmed by loss of recognition with the phospho dead T257A mutant.