Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Local translation in nuclear condensate amyloid bodies.

doi: 10.1073/pnas.2014457118

Figure Lengend Snippet: Fig. 1. Physiological stressors activate local nuclear protein synthesis. (A) Puromycin immunofluorescence in intact acidotic or thermal-stressed cells; 100:1 anisomycin competition abolishes signal. (B) In vitro puromycylation schematic. Cells are permeabilized on coverslips with 0.1% Nonidet P-40 and washed to complete cytosolic removal before incubation with 6′FAM-puromycin or puromycin at 4 °C. (C) FAM-puromycin signal in permeabilized thermal-stressed cells lacking cytosol; 100:1 anisomycin competition abolishes signal. Signal is observed in entire cellular population of thermal-stressed cells. (D) Competition for A-site–binding assays with anisomycin eliminate FAM-puromycin signal in thermal-stressed and acidotic cells. (E) Western blot analysis of in vitro pur- omycylation in cytosolic (1:5 diluted) and nuclear fractions. Bio-Rad precision plus protein ladder included. (F) Complementation assay shows no accumulation of soluble puromycylated proteins in heat-shocked cells. (Scale bars: 5 μm.) Pixel intensity values depicted are ×104. Error bars represent SEM. *P < 0.05.

Article Snippet: Bio-Rad precision plus protein ladder included. (F) Complementation assay shows no accumulation of soluble puromycylated proteins in heat-shocked cells. (Scale bars: 5 μm.)

Techniques: Immunofluorescence, In Vitro, Incubation, Binding Assay, Western Blot