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Image Search Results
Journal: Molecular Biology of the Cell
Article Title: SARM1 and TRAF6 bind to and stabilize PINK1 on depolarized mitochondria
doi: 10.1091/mbc.E13-01-0016
Figure Lengend Snippet: SARM1 binds to and recruits TRAF2/6 into PINK1 complexes. (A, B) Cell lysates from HEK293 cells cotransfected with vectors expressing TRAF6/2-Myc and SARM1-Flag, respectively, were immunoprecipitated with anti-Flag antibody and analyzed by Western blotting. (C) Cell lysates from HEK293 cells cotransfected with vectors expressing PINK1-HA and/or SARM1-Flag were immunoprecipitated with anti-Flag antibody and analyzed by Western blotting. Endogenous TRAF2 and TRAF6 were recovered in the immune complex of PINK1–SARM1.
Article Snippet: The antibodies used were as follows: rabbit polyclonal antibody against PINK1 (BC100-494; Novus Biologicals, Littleton, CO); rabbit polyclonal antibody against PINK1 raised in our previous study ( Murata et al. , 2011a );
Techniques: Expressing, Immunoprecipitation, Western Blot
Journal: Molecular Biology of the Cell
Article Title: SARM1 and TRAF6 bind to and stabilize PINK1 on depolarized mitochondria
doi: 10.1091/mbc.E13-01-0016
Figure Lengend Snippet: SARM1 promotes TRAF6-mediated ubiquitination of PINK1. (A) Ubiquitination of PINK1 by TRAF6 in cells. Cell lysates from SH-SY5Y cells transfected with PINK1-HA and Flag-ubiquitin (Ub) along with TRAF2-Myc or TRAF6-Myc were immunoprecipitated with anti-HA agarose and analyzed by Western blotting. (B) An in vitro ubiquitination assay was performed by incubation of purified GST-PINK1 with Flag-tagged SARM1, Myc-tagged TRAF2, or Myc-tagged TRAF6 in the presence of E1, E2 (UbcH13/Uev1a), and ubiquitin. Ubiquitination of PINK1 was detected by pull down with glutathione-agarose beads, followed by Western blot analysis for ubiquitin. (C) SARM1 promotes TRAF6-mediated PINK1 ubiquitination in SH-SY5Y cells. SH-SY5Y cells were transfected with designated constructs. Levels of PINK1 ubiquitination were determined by immunoprecipitation with anti-HA agarose, followed by Western blot analysis. (D) Ubiquitination levels are reduced in PINK1 mutants. The experiment was performed under conditions similar to those in C.
Article Snippet: The antibodies used were as follows: rabbit polyclonal antibody against PINK1 (BC100-494; Novus Biologicals, Littleton, CO); rabbit polyclonal antibody against PINK1 raised in our previous study ( Murata et al. , 2011a );
Techniques: Ubiquitin Proteomics, Transfection, Immunoprecipitation, Western Blot, In Vitro, Incubation, Purification, Construct
Journal: Molecular Biology of the Cell
Article Title: SARM1 and TRAF6 bind to and stabilize PINK1 on depolarized mitochondria
doi: 10.1091/mbc.E13-01-0016
Figure Lengend Snippet: PINK1 is ubiquitinated by TRAF6 upon impairment of mitochondria. (A) PINK1 is ubiquitinated under the condition of low membrane potential of mitochondria. SH-SY5Y cells were transfected with designated constructs for 48 h, followed by treatment with 10 μM CCCP for 6 h. Ubiquitination of PINK1 was determined by immunoprecipitation with anti-HA agarose, followed by Western blot analysis. (B) SH-SY5Y cells were treated with 10 μM CCCP for 6 h. Ubiquitination of endogenous PINK1 was determined by immunoprecipitation with anti-PINK1 antibody, followed by Western blot analysis. K63-linked ubiquitination of PINK1 was detected using biotinylated K63-TUBE1 and streptavidin-HRP. Arrowheads indicate immunoprecipitated PINK1. (C, D) Overexpression of dominant-negative TRAF6 destabilizes exogenous and endogenous PINK1 as shown in C and D, respectively. The experiment was performed under conditions similar to those in A. (E) Knockdown of TRAF6 inhibits stabilization of endogenous PINK1. SH-SY5Y cells were transfected with designated siRNAs for 72 h, followed by treatment with 10 μM CCCP for 6 h.
Article Snippet: The antibodies used were as follows: rabbit polyclonal antibody against PINK1 (BC100-494; Novus Biologicals, Littleton, CO); rabbit polyclonal antibody against PINK1 raised in our previous study ( Murata et al. , 2011a );
Techniques: Membrane, Transfection, Construct, Ubiquitin Proteomics, Immunoprecipitation, Western Blot, Over Expression, Dominant Negative Mutation, Knockdown
Journal: Molecular Biology of the Cell
Article Title: SARM1 and TRAF6 bind to and stabilize PINK1 on depolarized mitochondria
doi: 10.1091/mbc.E13-01-0016
Figure Lengend Snippet: TRAF6 ubiquitinates PINK1 at Lys433. SH-SY5Y cells were transfected with designated constructs along with C-terminal HA-tagged PINK1 wild type or mutants with different lysine-to-arginine mutations for 48 h. (A) Ubiquitination of PINK1 was determined by immunoprecipitation with anti-HA agarose, followed by Western blot analysis. (B) The experiment was performed under conditions similar to those in A. For K433R, double amount of plasmid was used compared with that of PINK1-WT to attain the same protein levels of PINK1. (C, D) SH-SY5Y cells transfected with designated constructs were treated with 10 μM CCCP for 6 h (C) or 0.5 μM MG-132 for 12 h (D).
Article Snippet: The antibodies used were as follows: rabbit polyclonal antibody against PINK1 (BC100-494; Novus Biologicals, Littleton, CO); rabbit polyclonal antibody against PINK1 raised in our previous study ( Murata et al. , 2011a );
Techniques: Transfection, Construct, Ubiquitin Proteomics, Immunoprecipitation, Western Blot, Plasmid Preparation
Journal: Molecular Biology of the Cell
Article Title: SARM1 and TRAF6 bind to and stabilize PINK1 on depolarized mitochondria
doi: 10.1091/mbc.E13-01-0016
Figure Lengend Snippet: SARM1 and TRAF6 promote the translocation of PINK1 to the outer membrane of mitochondria. (A) SH-SY5Y cells were transfected with SARM1-His for 48 h, followed by treatment with 10 μM CCCP for 2 h. The cell lysates were immunoprecipitated with anti-Tom20 antibody or control mouse IgG, followed by Western blot analysis. (B) SARM1 and TRAF6 promote the association of PINK1 with Tom20. SH-SY5Y cells were transfected with designated constructs for 48 h and analyzed under the conditions similar to those in A. Arrowhead indicates immunoprecipitated PINK1. (C) PINK1-WT but not OPA3-PINK1 interacts with SARM1. Cell lysates from SH-SY5Y cells transfected with SARM1-His along with PINK1-WT-HA or OPA3-PINK1-HA (PINK1 1-110 fused to the N-terminal mitochondrial anchor of OPA3 at amino acid positions 1–30) were immunoprecipitated with anti-HA agarose and analyzed by Western blotting.
Article Snippet: The antibodies used were as follows: rabbit polyclonal antibody against PINK1 (BC100-494; Novus Biologicals, Littleton, CO); rabbit polyclonal antibody against PINK1 raised in our previous study ( Murata et al. , 2011a );
Techniques: Translocation Assay, Membrane, Transfection, Immunoprecipitation, Control, Western Blot, Construct
Journal: Molecular Biology of the Cell
Article Title: SARM1 and TRAF6 bind to and stabilize PINK1 on depolarized mitochondria
doi: 10.1091/mbc.E13-01-0016
Figure Lengend Snippet: SARM1 and TRAF6 are required for recruitment of parkin to damaged mitochondria. SH-SY5Y (A) and HeLa cells (B) were transfected with Flag-parkin for 48 h, followed by treatment with 10 μM CCCP for 6 h and then immunostained with the indicated antibodies. (C–F) HeLa cells were transfected with designated constructs for 48 h. The number of cells with parkin-positive mitochondria among the parkin-expressing cells. Three independent experiments were performed, and at least 100 cells were counted for each experiment. Error bars represent mean ± SD. *Significantly different from the control group ( p < 0.01); ns, not significant. (C) Cells were transfected with PINK1 variants. (D) Effect of cotransfection with SARM1 and TRAF6. (E) Effect of knockdown of endogenous PINK1, SARM1, TRAF2, and TRAF6. (F) A rescue experiment for down-regulated endogenous proteins with overexpression of the respective proteins.
Article Snippet: The antibodies used were as follows: rabbit polyclonal antibody against PINK1 (BC100-494; Novus Biologicals, Littleton, CO); rabbit polyclonal antibody against PINK1 raised in our previous study ( Murata et al. , 2011a );
Techniques: Transfection, Construct, Expressing, Control, Cotransfection, Knockdown, Over Expression