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Results: 8

1.
Fig. 8

Fig. 8. A proposed mechanistic scheme showing the effect of CCS on protein aggregation and degradation pathways. From: 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.

The scheme illustrates the CCS-dependent and independent degradation pathways inhibited by 3-MA, a known specific inhibitor for macroautophagy pathway, or epoxo, an established proteosome inhibitor, respectively.

Ha Kun Kim, et al. Arch Biochem Biophys. ;509(2):177-185.
2.
Fig. 2

Fig. 2. Co-expression of CCS reduced the nonionic detergent insoluble fraction of FALS A4V and G85R SOD1 mutants. From: 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.

As described in Materials and Methods, “PBS-soluble fraction” (lane 1), “nonionic detergent-soluble fraction” (lane 2), and “nonionic detergent-insoluble fractions” (lane 3) were obtained and analyzed. Proteins (5 ug) 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.

Ha Kun Kim, et al. Arch Biochem Biophys. ;509(2):177-185.
3.
Fig. 3

Fig. 3. Effects of co-expressing WT and CCS mutant on the activity and aggregate of SOD1. From: 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.

(A) WT or A4V SOD1 was expressed in AAV293 cells with WT CCS, mutant CCS (C243/245S), or without CCS by transient transfection. The activity of SOD1 was detected in In-gel assay as described in Materials and Methods, and shows a dark yellow color. (B) Fractionated proteins obtained from AAV293 cells co-expressed with WT or mutant CCS or without CCS co-expression were analyzed on SDS-PAGE. S, nonionic detergent-soluble; I, nonionic detergent-insoluble; - These data represent a typical set of three independent experiments.

Ha Kun Kim, et al. Arch Biochem Biophys. ;509(2):177-185.
4.
Fig. 6

Fig. 6. Overexpressed CCS prevented the mitochondrial accumulation of A4V and G85R. From: 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.

AAV 293 cells were transiently transfected with A4V or G85R mutant expression vectors with or without CCS co-expression. Mitochondrial and cytosolic fractions were prepared as described in Materials and Methods and the proteins were separated by SDS-PAGE (A) and native gel (B). Mitochondrial Hsp70 (mtHsp70), α-tubulin, and mitochondrial SOD2 (for native gel) were used as marker proteins for mitochondrial and cytosolic fractions. An immunoblot membrane was probed with anti-SOD1, anti-CCS, anti-mtHsp70 and anti-α-tubulin antibodies. The activity of SOD1 was detected in In-gel assay (B) as described in Materials and Methods, and shows a dark yellow color. These data represent results obtained from three independent experiments.

Ha Kun Kim, et al. Arch Biochem Biophys. ;509(2):177-185.
5.
Fig. 7

Fig. 7. H2O2 induces A4V accumulation in mitochondria of AAV 293 cells co-express with A4V and CCS. From: 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.

AAV 293 cells were transiently transfected with A4V mutant expression vectors with or without CCS co-expression. One day after transfection, the cells were treated with 200 μM H2O2 for 15 min. (A) Mitochondrial and cytosolic fractions were prepared and analyzed by Western blotting as described previously. (B) Relative mitochondrial levels of A4V in CCS co-expressed cells in the presence or absence of H2O2 were plotted relative to those observed without CCS co-expression. Values are means ± SEM (n = 5). *p <0.05 vs. control.

Ha Kun Kim, et al. Arch Biochem Biophys. ;509(2):177-185.
6.
Fig. 1

Fig. 1. Effect of simultaneous expression of FALS hSOD1 and hCCS on SOD1 containing HMWS in AAV 293 cells. From: 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.

(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.

Ha Kun Kim, et al. Arch Biochem Biophys. ;509(2):177-185.
7.
Fig. 4

Fig. 4. Effects of proteasome inhibitor, epoxomicin, and macroautophagy inhibitor, 3-methyladenine, on the accumulation of FALS mutant SOD1 and CCS levels. From: 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.

FALS SOD1 mutants were expressed in HEK293 cells with or without CCS by transient transfection. Twenty-four hours after transfection, media were replaced with fresh ones that contained the indicated inhibitors and incubated for another 12 hrs or 24 hrs. Fractionated proteins (5 ug) were loaded onto each lane and subjected to SDS-PAGE in the presence of reducing agent in 10-20% Tris-Glycine gel. (A) Level of A4V and CCS monitored by Western blot after treatment with 100 nM epoxomicin for 12 hrs or 24 hrs as indicated. (B) A4V and CCS monitored by Western blot after treatment with 5 mM 3-methyladenine (3-MA) for 24 hrs. (C) G85R and CCS monitored by Western blot after treatment with 100 nM epoxomicin or 5 mM 3-MA for 24 hrs. (D) The relative change in SOD1 and CCS levels due to epoxomicin or 3-MA treatments are expressed in fold increase relative to control in each group. The histogram represents means ± SEM (n ≥ 5) of fold increase relative to that observed in the absence of indicated inhibitor. S, nonionic detergent-soluble: I, nonionic detergent-insoluble; -, control; E, epoxomicin; M, 3-MA; Endo, endogeneous SOD1. *p<0.05; **p <0.01 vs. control.

Ha Kun Kim, et al. Arch Biochem Biophys. ;509(2):177-185.
8.
Fig. 5

Fig. 5. Complexes formed between G85R and CCS, and between SOD1 and Hsp-70 detected by co-immunoprecipitation. From: 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.

AAV 293 cells were transiently transfected with (A, 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 hr 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 (10,000×g 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,C) were added to a supernatant aliquot containing 500 μg of cellular proteins and incubated for 1 hr at 4°C. To isolate the SOD1 antibody conjugated proteins (A) or the Hsp70 antibody conjugated proteins (B,C), Protein A-conjugated agarose beads (Pierce) were added and the mixture incubated for another 1 hr 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.

Ha Kun Kim, et al. Arch Biochem Biophys. ;509(2):177-185.

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