Fig 1: PINK1 is required for Parkin translocation but interaction between Beclin1 and Parkin is PINK1 independent. (A) HEK293T cells were transfected with scrambled siRNA and siRNA targeting SESN2 followed by treatment with 10 µM CCCP for 3 h. Total cell lysates were co-immunoprecipitated using anti-PINK1 and anti-Parkin antibodies, to find the variation in interaction between endogenous PINK1 and Parkin. Interaction of PINK1 with Parkin is less prominent on downregulation of SESN2. (B) Effect of PINK1 on mitochondrial translocation of Parkin was assessed by transfecting cells with scrambled siRNA and PINK1 siRNA followed by treatment with 10 µM CCCP for 3 h. Cellular sub-fractionation was done and mitochondrial fraction was subjected to immunoblotting. TOMM20, mitochondrial marker. 1-DMSO, 2-CCCP. Note that PINK1 knockdown abolishes the presence of Parkin. (C) To check the mitochondrial stabilization of PINK1 in SESN2 knockdown cells, cells were treated with DMSO (vehicle) and CCCP for 3 h and western blot analysis was carried out in isolated mitochondrial fractions. VDAC1, mitochondrial marker and TUBB/ß-Tubulin, cytosolic marker. Levels of SESN2 in PINK1 knockdown cells were analysed with western blots in whole cell lysates. 1-DMSO, 2-CCCP. Note: downregulation of SESN2 or PINK1 was unable to decline stability of each other. (D) Wild-type (WT) and PINK1 knockdown cells were treated with DMSO (vehicle) and CCCP for 3 h. Cytosolic fractions were isolated and subjected to co-IP assay using anti-Beclin1 and anti-Parkin antibodies. Immunoblots were converted to gray and white scale using Adobe Photoshop CS6, with linear adjustment of exposure or contrast. Images of immunoblots were cropped for clarity, expanded view of Fig. 6D is available in supplementary figures.