Journal: Archives of biochemistry and biophysics

Article Title: Effect of CCS on the accumulation of FALS SOD1 mutant-containing aggregates and on mitochondrial translocation of SOD1 mutants: implication of a free radical hypothesis.

doi: 10.1016/j.abb.2011.02.014

Figure Lengend Snippet: Fig. 1. Effect of simultaneous expression of FALS hSOD1 and hCCS on SOD1- containing HMWS in AAV293 cells. (A) Schematic representation of plasmid vectors used to produce dicistronic mRNA for hSOD1 and hCCS co-expression. B, BamHI; X, XhoI; N, NcoI; K, KpnI; RI, EcoRI. (B) Detection of mutant SOD1 with or without CCS revealed with reducing gel (see Materials and methods). Top panel shows a hSOD1 blot visualized by ECL chemiluminescence. Asterisks depict the high molecular- weight species. The CCS proteins were also detected by the sheep polyclonal hSOD1 (574597) antibody due to the high homology of the two proteins. Middle panel shows the identical SOD1 blot as the top panel, except with shorter exposure time. Lower panel represents a CCS blot. This figure shows typical results obtained with three independent experiments.

Article Snippet: The primary antibodies used were as follows: sheep polyclonal anti-human Cu,Zn SOD1 (574597) (1:2000; Calbiochem, La Jolla, CA, USA), rabbit polyclonal anti-human Cu,Zn SOD1 (RDI-SODabRx) (1:5000; Fitzgerald, Concord, MA, USA), mouse monoclonal anti-mitochondrial Hsp70 (MN3-028) (1:5000; Affinity Bioreagents, Golden, CO), mouse monoclonal anti-human CCS (2A1) (1:5000; Abcam, Cambridge, MA, USA), mouse monoclonal anti-actin (1:10,000; Sigma), and mouse monoclonal anti-Hsp70 (W27) (1:1000; Santa Cruz Biotechnology, Inc., Santa Cruz, CA).

Techniques: Expressing, Plasmid Preparation, Mutagenesis, High Molecular Weight

Journal: Archives of biochemistry and biophysics

Article Title: Effect of CCS on the accumulation of FALS SOD1 mutant-containing aggregates and on mitochondrial translocation of SOD1 mutants: implication of a free radical hypothesis.

doi: 10.1016/j.abb.2011.02.014

Figure Lengend Snippet: Fig. 2. Co-expression of CCS reduced the non-ionic detergent-insoluble fraction of FALS A4V and G85R SOD1 mutants. As described in Materials and methods, ‘‘PBS- soluble fraction’’ (lane 1), ‘‘non-ionic detergent-soluble fraction’’ (lane 2), and ‘‘non- ionic detergent-insoluble fractions’’ (lane 3) were obtained and analyzed. Proteins (5 lg) were loaded onto 10–20% gradient gels. Blots were obtained with rabbit polyclonal antibody (RDI-SODabRx) to Cu,Zn SOD1, or with monoclonal antibody to CCS, and visualized by ECL chemiluminesence.

Article Snippet: The primary antibodies used were as follows: sheep polyclonal anti-human Cu,Zn SOD1 (574597) (1:2000; Calbiochem, La Jolla, CA, USA), rabbit polyclonal anti-human Cu,Zn SOD1 (RDI-SODabRx) (1:5000; Fitzgerald, Concord, MA, USA), mouse monoclonal anti-mitochondrial Hsp70 (MN3-028) (1:5000; Affinity Bioreagents, Golden, CO), mouse monoclonal anti-human CCS (2A1) (1:5000; Abcam, Cambridge, MA, USA), mouse monoclonal anti-actin (1:10,000; Sigma), and mouse monoclonal anti-Hsp70 (W27) (1:1000; Santa Cruz Biotechnology, Inc., Santa Cruz, CA).

Techniques: Expressing

Journal: Archives of biochemistry and biophysics

Article Title: Effect of CCS on the accumulation of FALS SOD1 mutant-containing aggregates and on mitochondrial translocation of SOD1 mutants: implication of a free radical hypothesis.

doi: 10.1016/j.abb.2011.02.014

Figure Lengend Snippet: Fig. 5. Complexes formed between G85R and CCS, and between SOD1 and Hsp70 detected by co-immunoprecipitation. AAV293 cells were transiently transfected with (A and C) G85R-GFP or G85R-CCS vectors and with (B) FLAG-tagged WT SOD1 or A4V mutant expression vectors with or without CCS co-expression. At 48 h post- transfection, cells were lysed in lysis buffer (20 mM Hepes, 1% Triton X-100, 10% glycerol, pH 7.4) that contained protease inhibitor cocktail (Sigma). After brief sonication, cell lysates were centrifuged (10,000g for 5 min) and the supernatant was collected. Rabbit polyclonal antibody that does not cross-react with CCS, against SOD1 (RDI-SODabRx) (A) or mouse monoclonal antibody against Hsp70 (MN3-028) (B and C) were added to a supernatant aliquot containing 500 lg of cellular proteins and incubated for 1 h at 4 C. To isolate the SOD1 antibody conjugated proteins (A) or the Hsp70 antibody conjugated proteins (B and C), protein A-conjugated agarose beads (Pierce) were added and the mixture incubated for another 1 h at 4 C. After washing with wash buffer, the immunoprecipitates were recovered with 1 SDS sample buffer, separated by SDS–PAGE, and probed with rabbit anti-SOD1 (RDI-SODabRx), mouse monoclonal anti-CCS (2AI) and anti- Hsp70 antibodies (MN3-028) as indicated. IB, immunoblotting; IP, immunoprecipitation.

Article Snippet: The primary antibodies used were as follows: sheep polyclonal anti-human Cu,Zn SOD1 (574597) (1:2000; Calbiochem, La Jolla, CA, USA), rabbit polyclonal anti-human Cu,Zn SOD1 (RDI-SODabRx) (1:5000; Fitzgerald, Concord, MA, USA), mouse monoclonal anti-mitochondrial Hsp70 (MN3-028) (1:5000; Affinity Bioreagents, Golden, CO), mouse monoclonal anti-human CCS (2A1) (1:5000; Abcam, Cambridge, MA, USA), mouse monoclonal anti-actin (1:10,000; Sigma), and mouse monoclonal anti-Hsp70 (W27) (1:1000; Santa Cruz Biotechnology, Inc., Santa Cruz, CA).

Techniques: Immunoprecipitation, Transfection, Mutagenesis, Expressing, Lysis, Protease Inhibitor, Sonication, Incubation, SDS Page, Western Blot