Journal: G3: Genes|Genomes|Genetics
Article Title: Heparan Sulfate Structure Affects Autophagy, Lifespan, Responses to Oxidative Stress, and Cell Degeneration in Drosophila parkin Mutants
Figure Lengend Snippet: Reduction of heparan sulfate biosynthetic function prevents increases of ubiquitin-modified protein in the brains of ROS exposed animals. Accumulation of insoluble ubiquitinated proteins (IUPs) was examined in the triton-X100 insoluble fraction of total head proteins. UAS-mCherry RNAi, an shRNAi with no predicted targets in the Drosophila genome, was used as a control. All plus/minus oxidant pairs are from the same blot. All lanes are from the same experiment, with the same standard sample loaded on all gels from this one experiment for standardization (see Supplemental Figure 3 for original blots). Overexpression of Atg8a ( UAS-Atg8a ) was used as a positive control for enhancement of autophagy. The UAS-constructs were expressed under control of elav -GAL4 with UAS-dcrII to enhance RNAi efficacy. Lanes from anti-ubiquitin stained membranes are shown in control vs. oxidant-exposed pairs. The ratio depicted is the average density of anti-ubiquitin staining, normalized to loading, in samples with peroxide (+) divided by samples without peroxide (-). Two different sfl RNAi lines were tested. Two additional replicate experiments gave average peroxide/no peroxide ratios of 1.23 for control, and 0.8 for sfl RNAi-HMS . Two replicates of ttv RNAi also showed lower levels of ubiquitin-insoluble material upon peroxide exposure (ratio of 0.7).
Article Snippet: Typically, 30-60 adult heads were homogenized by ceramic bead agitation using the Bead Ruptor 24 in 150ul of Triton-X100 extraction buffer (1% Triton-X100, 1x PBS, 1X protease inhibitor cocktail Complete [Roche, 10184600]).
Techniques: Modification, Over Expression, Positive Control, Construct, Staining