Journal: Autophagy

Article Title: Isorhynchophylline, a natural alkaloid, promotes the degradation of alpha-synuclein in neuronal cells via inducing autophagy.

doi: 10.4161/auto.8.1.18313

Figure Lengend Snippet: Figure 4. IsoRhy induced autophagy in L3 Drosophila larvae fat body. (A) Ninety six hours after egg laying, the L3 larvae were collected and fed on culture medium containing 0.2 mg/ml of IsoRhy for 6 h and fat bodies were isolated for LysoTracker staining. Images were captured using a fluorescence microscope. As positive control, L3 larvae were either fed with 5 mM of rapamycin for 24 h or starved for 3 h to induce autophagy. (B) Quantification of lysotracker-positive spots in each group. Data were presented as the mean ± SEM of one representative experiment from three independent experiments. (***p , 0.001, one-way ANOVA for multiple comparison and Tukey’s test as post hoc test).

Article Snippet: The instant Drosophila food (173212) was purchased from Carolina Biological Supply Company.

Techniques: Isolation, Staining, Fluorescence, Microscopy, Positive Control, Comparison

Journal: PLoS Biology

Article Title: The mitochondrial transporter SLC25A25 links ciliary TRPP2 signaling and cellular metabolism

doi: 10.1371/journal.pbio.2005651

Figure Lengend Snippet: (A) Purified SLC25A25 (5 μg) was separated by SDS-PAGE and visualized by Coomassie staining. (B) Thermostability profile (top) and its corresponding first derivative (bottom) of SLC25A25. Assays were performed in triplicate with the addition CaCl 2 (orange line) or without (blue line). (C) Uptake assays of [ 14 C]-labeled Mg-ATP into proteoliposomes with the addition CaCl 2 (orange line) and without (blue line). The error bars represent the standard deviation of 4 replicates, and the uptake curves were fitted with a one-phase association curve (r-squared > 0.99). For numerical values, see . AU, arbitrary unit; SLC25A25, solute carrier 25 A 25.

Article Snippet: Proteins were separated by using 4%–12% TruPAGE precast gels SDS-PAGE gels (Merck KGaA) loaded with 5 μg of protein at a 3:1 mix of sample to loading buffer and stained using Instant Blue Coomassie according to manufacturer’s instructions (Merck KGaA).

Techniques: Purification, SDS Page, Staining, Labeling, Standard Deviation

Journal: The Journal of Biological Chemistry

Article Title: The ubiquitin-like modifier FAT10 stimulates the activity of deubiquitylating enzyme OTUB1

doi: 10.1074/jbc.RA118.005406

Figure Lengend Snippet: FAT10 and USE1 enhance the cleavage activity of OTUB1 in vitro. A, recombinant Lys-48 or Lys-63 DiUb (5 μm) was incubated with tagless OTUB1 (5 μm) for the indicated time periods at 37 °C, and reactions were stopped by the addition of gel sample buffer supplemented with 4% 2-mercaptoethanol. Proteins were separated on 4–12% NuPAGE bis-Tris gels with subsequent colloidal Coomassie staining. B, quantitative analysis of AMC fluorescence (ex. 360 nm, em. 465 nm) of ubiquitin-AMC (5 μm) incubated with His-tagged or tagless OTUB1 WT or C91S (all 10 μm) for the indicated time periods at 30 °C. C, silver-stained gel of recombinant Lys-48–linked DiUb (5 μm) incubated for the indicated time periods with tagless OTUB1 (5 μm) and tagless FAT10, UbcH5B-His, and His-USE1 (all 10 μm) at 37 °C. The reactions were stopped by the addition of gel sample buffer supplemented with 4% 2-mercaptoethanol. D, densitometric analysis of cleaved Lys-48–linked DiUb by OTUB1 in the presence of FAT10, USE1, and UbcH5B. The DiUb protein amount at time point 0 was set to 100%. E, enlarged focus on the first 10 min of the densitometric analysis in D. F, FRET-based analysis for the cleavage of internally quenched Lys-48 DiUb (400 nm) by OTUB1 WT or C91S (both 30 nm) in the absence or presence of FAT10, USE1, and UbcH5B (all 5 μm). One representative experiment of three experiments with similar outcomes is shown.

Article Snippet: Gels were stained with colloidal Coomassie (Instant Blue, Gentaur, Aachen, Germany) or silver stain (Thermo Scientific).

Techniques: Activity Assay, In Vitro, Recombinant, Incubation, Staining, Fluorescence