Fig. 6.

Tom20/Tom40 and Tim23 complex are necessary for SHP2 translocation to mitochondrial matrix. a Predication of mitochondrial target sequence in SHP2 by PSORT II. b Immunoblot analysis of GFP localization in submitochondrial fractions from HEK293T cells which were transfected with GFP or RRWFH-GFP plasmid. c Immunoblot analysis of SHP2 localization in submitochondrial fractions from HEK293T cells which were transfected with SHP2-HA or SHP2-mut-HA (mitochondrial target sequence mutation, RRWFH mutated to AAWFH) plasmid followed by ATP treatment (5 mM, 30 min). d Immunofluorescence analysis mitochondrial localization of GFP-tagged RRWFH motif in HEK293T cells. Scale bar, 10 µm. e Immunofluorescence analysis mitochondrial localization of SHP2-HA or SHP2-mut-HA plasmid in HEK293T cells. Scale bar, 10 µm. f Co-immunoprecipitation (Co-IP) analysis of the interaction of SHP2 and ANT1 in HEK293T cells, which were transfected with ANT1-myc and SHP2-HA or SHP2-mut-HA. g Flow cytometry analysis of mitochondrial membrane potential by JC-1 staining in HEK293T cells which were transfected with SHP2-HA or SHP2-mut-HA plasmid followed by cccp treatment (20 µM, 1 h). h Immunoblot analysis of SHP2 in mitochondria after Tom20 or Tom40, or Tom70 sliencing. Endogenous Toms were separately knocked down by its corresponding shRNAs in THP-1 cells followed by ATP treatment (5 mM, 30 min), then mitochondria were isolated and incubated with 40 μM proteinase K (Pro K) for 30 min. Tom20 in mitochondrial outer membrane (MOM) and Tim23 in mitochondrial inner membrane (MIM) were used as control, respectively. i Immunoblot analysis of SHP2 expression in submitochondrial fractions from THP-1-derived macrophages treated with ATP (5 mM, 30 min) after Tim22 or Tim23 silencing. *P < 0.05 by Student’s t-test, NS represents no significance. Data are representative of three independent experiments (mean and SEM of three independent samples in g)