Journal: PLoS ONE
Article Title: Characterization of Detergent-Insoluble Proteins in ALS Indicates a Causal Link between Nitrative Stress and Aggregation in Pathogenesis
doi: 10.1371/journal.pone.0008130
Figure Lengend Snippet: (A) Total TIF is the ratio of the amount of TIF to the total proteins extracted. Proteins were quantified in each condition by the BCA protein assay. Values are percentages of the untreated WT control and are the mean±SEM (n = 3). (B–G) The level of nitrotyrosine, nitrated HSP90 (HSP90 NT ), aconitase, HSC70, CypA and SOD1 were measured by dot blot analysis. The same amount of cellular TIF (3 µg) was loaded on the membrane and probed with the specific antibodies. Histograms represent the immunoreactivity normalized to the actual amount of protein loaded, as detected after Red Ponceau staining, multiplied by the total TIF isolated for each condition. Values are percentages of the untreated WT control and are the mean±SEM (n = 3). *, significantly different from untreated G93A controls ( p <0.05); **, significantly different from MG132-treated G93A samples ( p <0.05); ***, significantly different from MG132-treated WT samples ( p <0.05), as assessed by one-way ANOVA followed by Newman-Keuls multiple comparison test. (H) Analysis of cell death by quantification of extracellular LDH activity. Histograms represent mean±SD of four replicates. One-way ANOVA was followed by Newman-Keuls multiple comparison test. *, p <0.05.
Article Snippet: One 2D gel was stained with SYPRO® Ruby protein gel stain (Invitrogen) and the other was transferred onto PVDF membrane (Millipore) and probed overnight at 4°C with anti-nitrotyrosine rabbit polyclonal antibody, provided by A.G. Estevez, or the anti-nitrotyrosine mouse monoclonal antibody (clone HM.11; HyCult Biotechnology).
Techniques: Bicinchoninic Acid Protein Assay, Dot Blot, Staining, Isolation, Activity Assay