Journal: Molecular neurobiology

Article Title: Potential Effect of S -Nitrosylated Protein Disulfide Isomerase on Mutant SOD1 Aggregation and Neuronal Cell Death in Amyotrophic Lateral Sclerosis

doi: 10.1007/s12035-013-8562-z

Figure Lengend Snippet: PDI decreases SOD1 monomers as well as multimers. a Lysates from HEK293A cells transfected with SOD1 and PDI were examined by Western blotting under nonreducing conditions. b, c PDI decreased the relative level of mSOD1 (G93A) protein (shown on Y-axis), and the decrease was observed for both monomers (b) and multimers (c). However, the PDI-induced decrease in SOD1 levels was accentuated for SOD1 monomers of both the wild-type and mutant (SOD1: WT wild-type, G93A G93A mutant, Moc mock-transfected). Values are mean + SEM, n =4; *P <0.01, **P <0.001

Article Snippet: Cell Culture and ER Stress HEK293A cells and NSC-34 cells (CELLutions Biosystems) stably expressing SOD1 WT or SOD1 G93A were grown in Dulbecco’s modified Eagle’s medium (DMEM, Hyclone, Salt Lake City, UT, USA) containing 10 % heat-inactivated fetal bovine serum (Hyclone), 2 % penicillin/streptomycin, and 2 mM glutamine in a humidified incubator at 37 °C under 5 % CO 2 .

Techniques: Transfection, Western Blot, Mutagenesis

Journal: Molecular neurobiology

Article Title: Potential Effect of S -Nitrosylated Protein Disulfide Isomerase on Mutant SOD1 Aggregation and Neuronal Cell Death in Amyotrophic Lateral Sclerosis

doi: 10.1007/s12035-013-8562-z

Figure Lengend Snippet: Overexpression of PDI decreases SOD1 monomers as well as multimers. a, b HEK293A cells were transfected with SOD1 and wild-type (WT) PDI or mutant (MT) PDI, and the change in SOD1 protein was examined by immunoblotting under nonreducing (a) and reducing (b) conditions. In mutant PDI, the four cysteine residues were mutated to serine (C36S, C39S, C383S, and C386S). Expression of PDI decreased total SOD1 protein. c Media of NSC-34 cells expressing SOD1 and/or PDI was monitored for secreted SOD1. SOD1 in the media was seen in the multimer form and was not significantly different in regardless of the presence of PDI. Values are mean + SEM, n =4; *P <0.01, **P <0.001

Article Snippet: Cell Culture and ER Stress HEK293A cells and NSC-34 cells (CELLutions Biosystems) stably expressing SOD1 WT or SOD1 G93A were grown in Dulbecco’s modified Eagle’s medium (DMEM, Hyclone, Salt Lake City, UT, USA) containing 10 % heat-inactivated fetal bovine serum (Hyclone), 2 % penicillin/streptomycin, and 2 mM glutamine in a humidified incubator at 37 °C under 5 % CO 2 .

Techniques: Over Expression, Transfection, Mutagenesis, Western Blot, Expressing

Journal: Molecular neurobiology

Article Title: Potential Effect of S -Nitrosylated Protein Disulfide Isomerase on Mutant SOD1 Aggregation and Neuronal Cell Death in Amyotrophic Lateral Sclerosis

doi: 10.1007/s12035-013-8562-z

Figure Lengend Snippet: Filter trap assay. a Overexpression of PDI or mutant PDI decreased the quantity of mSOD1 accumulating on the filter. b SNOC significantly attenuated the ability of PDI to decrease filter-trapped SOD1. Quantification from n =4 experiments shown below sample filter sets. Values are mean + SEM; *P <0.01

Article Snippet: Cell Culture and ER Stress HEK293A cells and NSC-34 cells (CELLutions Biosystems) stably expressing SOD1 WT or SOD1 G93A were grown in Dulbecco’s modified Eagle’s medium (DMEM, Hyclone, Salt Lake City, UT, USA) containing 10 % heat-inactivated fetal bovine serum (Hyclone), 2 % penicillin/streptomycin, and 2 mM glutamine in a humidified incubator at 37 °C under 5 % CO 2 .

Techniques: TRAP Assay, Over Expression, Mutagenesis

Journal: Molecular neurobiology

Article Title: Potential Effect of S -Nitrosylated Protein Disulfide Isomerase on Mutant SOD1 Aggregation and Neuronal Cell Death in Amyotrophic Lateral Sclerosis

doi: 10.1007/s12035-013-8562-z

Figure Lengend Snippet: Co-localization of overexpressed wild-type SOD1, SOD1 (G93A) mutant, and PDI in NSC-34 cells. Cells receiving the PDI construct were stained immunocytochemically 48 h after transfection. Images of wild-type SOD1 (green), SOD1 (G93A) mutant (green), and PDI (red) and merge were monitored under confocal microscopy. Wild-type SOD1 stained diffusely inside of cells, consistent with its previously reported cytosolic distribution (a), while mSOD1 did not stain the nucleus, indicating its nuclear-sparing pattern of distribution (b). Wild-type and m SOD1 coprecipitate with PDI and induce PDI expression in NSC-34 cells. The anti-SOD1 antibody coprecipitated PDI, and the anti-PDI antibody coprecipitated SOD1 in NSC-34 cells transfected with wild-type SOD1 or m SOD1. SOD1 thus interacts with PDI in NSC-34 cells transfected with wild-type SOD1 or m SOD1 (c)

Article Snippet: Cell Culture and ER Stress HEK293A cells and NSC-34 cells (CELLutions Biosystems) stably expressing SOD1 WT or SOD1 G93A were grown in Dulbecco’s modified Eagle’s medium (DMEM, Hyclone, Salt Lake City, UT, USA) containing 10 % heat-inactivated fetal bovine serum (Hyclone), 2 % penicillin/streptomycin, and 2 mM glutamine in a humidified incubator at 37 °C under 5 % CO 2 .

Techniques: Mutagenesis, Construct, Staining, Transfection, Confocal Microscopy, Expressing

Journal: Molecular neurobiology

Article Title: Potential Effect of S -Nitrosylated Protein Disulfide Isomerase on Mutant SOD1 Aggregation and Neuronal Cell Death in Amyotrophic Lateral Sclerosis

doi: 10.1007/s12035-013-8562-z

Figure Lengend Snippet: S-Nitrosylation of PDI (forming SNO–PDI) in vitro and in vivo. Biotin switch shows SNO–PDI and standard immunoblot, total PDI. a Levels of total PDI and SNO–PDI increased in mSOD1 (G93A)-transfected NSC-34 cells in the presence of thapsigargin or SNOC. b, c SNO–PDI was found in the spinal cords of SOD1 (G93A) mice and human patients with sporadic ALS but not in normal controls by biotin switch assay. Immunoblotting of input samples showed that total PDI was also increased in transgenic mSOD1 (G93A) mice (b) and ALS patients (c). d Increased ratio of SNO–PDI (by biotin switch) to total PDI (by immunoblot) in the in vitro conditions shown in (a), in mSOD1 (G93A) mice, and in sporadic ALS patient spinal cord. Values are mean + SEM, n =4, n =2 (normal control). *P <0.01, **P <0.001

Article Snippet: Cell Culture and ER Stress HEK293A cells and NSC-34 cells (CELLutions Biosystems) stably expressing SOD1 WT or SOD1 G93A were grown in Dulbecco’s modified Eagle’s medium (DMEM, Hyclone, Salt Lake City, UT, USA) containing 10 % heat-inactivated fetal bovine serum (Hyclone), 2 % penicillin/streptomycin, and 2 mM glutamine in a humidified incubator at 37 °C under 5 % CO 2 .

Techniques: In Vitro, In Vivo, Western Blot, Transfection, Biotin Switch Assay, Transgenic Assay, Control