Journal: Aging Cell
Article Title: “Protein aggregates” contain RNA and DNA, entrapped by misfolded proteins but largely rescued by slowing translational elongation
Figure Lengend Snippet: Effects of EEF2 knockdown on the composition of aggregates in SY5Y‐APP Sw cells. Results shown in each panel comprise data from 3 independent cell expansions treated with shRNA constructs targeting EEF2, or 2 scrambled RNAs for controls. Replicate experiments produced similar results. (A, B), Western‐blot quantitation of EEF2 knockdown efficacy, evaluated by EEF2 protein; efficacies of individual shRNAs (constructs a, b, c in Methods) are superimposed in B. (C, D), total RNA fragments in aggregates, quantified by gel staining with SYBR Gold. (E, F) Total DNA fragments in aggregates, quantified by ethidium bromide fluorescence. (G, H) Total aggregate protein, quantified by staining with SYPRO Ruby. p values shown here are based on 2‐tailed heteroscedastic t tests, for 3 – 4 experiments, combining data from shRNAs a – c
Article Snippet: These consisted of (1) separation of RNA and DNA fragments using a Qiagen AllPrep DNA/RNA extraction kit according to the manufacturer's protocol, with recovery assayed by absorbance at 260 nm; (2) separation of RNA and DNA fragments with the Qiagen kit, and quantitation by ethidium bromide and/or SYBR Gold after resolution by acrylamide gel electrophoresis; (3) selective enzymatic digestion with RNAse‐free DNAse (Thermo Fisher, CA) and assay by 260‐nm absorption (RNA directly; DNA by subtraction), and (4) using TRI Reagent (Molecular Research Ctr., TR118) to isolate RNA, DNA, and protein in a single protocol.
Techniques: shRNA, Construct, Produced, Western Blot, Quantitation Assay, Staining, Fluorescence