Journal: Journal of Biological Chemistry

Article Title: Cysteine 70 of Ankyrin-G Is S-Palmitoylated and Is Required for Function of Ankyrin-G in Membrane Domain Assembly

doi: 10.1074/jbc.m112.417501

Figure Lengend Snippet: FIGURE 7. C70A mutation abolishes AnkG function in lateral membrane biosynthesis. A, doxycycline-inducible AnkG shRNA MDCK cell line was generated using an engineered Tet-pLKO-mCherry vector. Two days of doxycycline induction could selectively knock down 90% of 200-kDa AnkG. (Blot is represent- ative of at least three independent experiments). B, XZ planes of lateral membrane under different conditions (green, AnkG; white, ZO-1, scale bar, 10 m). AnkG knockdown cells show a defect in lateral membrane height compared with luciferase-shRNA cells. WT AnkG, but not the C70A mutant, is able to restore the height of lateral membrane. C, quantitative results of the height of lateral membranes. The height was defined as the distance between the bottom of ZO-1 staining and the end of the lateral membrane. Mean lateral membrane height was 9.8 0.3 m for luciferase shRNA, 3.4 0.4 m for AnkG shRNA, 9.1 0.6 m for WT-AnkG rescue, and 4.1 0.5 m for rescue with C70A AnkG-GFP. Conditions were compared using one-way ANOVA followed by a Tukey post hoc test (p 0.001 for ANOVA, n 24–30/condition). *, p 0.05. Error bars, S.E.

Article Snippet: GraphPad Prism version 5 was used to analyze the data by fitting them to the one-phase association equation, y y0 Ae k t. Doxycycline-inducible shRNA Cell Lines and Rescue Experiments—The Tet-pLKO-puro vector was originally obtained from Addgene (Plasmid 21915).

Techniques: Mutagenesis, Membrane, shRNA, Generated, Plasmid Preparation, Knockdown, Luciferase, Staining

Journal: Nature

Article Title: Two Distinct RNase Activities of CRISPR-C2c2 Enable Guide RNA Processing and RNA Detection

doi: 10.1038/nature19802

Figure Lengend Snippet: a, Illustration of LbuC2c2 RNA detection approach using a quenched fluorescent RNA reporter. b, Quantification of fluorescence signal generated by LbuC2c2 after 30 min for varying concentrations of target RNA in the presence of human total RNA. RNase A shown as positive RNA degradation control. (mean ± s.d., n = 3) c,. Quantification of fluorescence signal generated by LbuC2c2 loaded with a β-actin targeting crRNA after 3h for varying amounts of human total RNA or bacterial total RNA (as a β-actin null negative control). (mean ± s.d., n = 3) d, Tandem pre-crRNA processing also enables RNA detection. (mean ± s.d., n = 3) e, Model of the Type VI CRISPR pathway highlighting both of C2c2’s ribonuclease activities.

Article Snippet: The percent cleavage was determined as the ratio of total banding intensity for all shorter products relative to the uncleaved band and normalized for background within each measured substrate using ImageQuant TL Software (GE Healthcare) and fit to a one phase exponential association using Prism (GraphPad). crRNA filter-binding assays Filter binding assays was carried out in RNA processing buffer (20 mM HEPES pH 6.8, 50 mM KCl, 5 mM MgCl 2 , 10 μg/mL BSA, 10 μg/mL yeast tRNA, 0.01% Igepal CA-630 and 5% glycerol).

Techniques: RNA Detection, Fluorescence, Generated, Negative Control, CRISPR