Fig. 5

Fig. 5

Tamm41 is required for the PARK2-PINK1 mitophagy process. a, b Elevated unstabilized PINK1 (50KD) in tamm41^−/− embryos. c, d Representative images (n = 50 cells per group) show that TAMM41 silencing affected CCCP induced HA-PARK2 (red) recruitment onto mitochondria (TOMM20, green) in AC16 cells. Scale bar: 20 μm. e, f Western blot assessment of mitochondrial-fractionated PARK2 levels in transfected AC16 cells. TAMM41 deficiency inhibited PARK2 mitochondrial accumulation elicited by CCCP addition. g, h Representative images (n = 50 cells per group) show that reducing TAMM41 expression impeded CCCP triggered PINK1-GFP mitochondrial stabilization (TOMM20, red). Scale bar: 20 μm. i Representative images (n = 10 per group) reveal that CCCP treatment induced HA-PARK2 (red) translocation onto mitochondria (TOM20, green) in tamm41^+/+ hearts (white arrows), but failed to do so in tamm41^−/− hearts. Scale bar: 20 μm. j Enhanced HA-PARK2 recruitment onto mitochondria in TAMM41-GFP overexpressed AC16 cells (white arrowhead) compared with cell without TAMM41 overexpression (asterisk). Scale bar: 20 μm. k, l TAMM41-GFP overexpression promoted PINK1 stabilization (60KD) in the AC16 cell line by western blot. m Co-immunoprecipitation assay demonstrates the interaction between TAMM41-GFP and PINK1-V5. Black horizontal lines indicate mean ± SD. Means ± SD are shown for three independent experiments. n.s, not significant, **P < 0.01; ***P < 0.001 (Student’s t-test)