Figure 3

Localization and FRET interactions of endogenous ARE binding proteins upon oxidative stress: P bodies and Stress Granules. ARE-BPs were detected via immunocytochemistry. Cells were treated with 0.5 mM sodium arsenite (SA) for 30 minutes. PBs were defined by detection of Hedls. The localization of each ARE-BP and Hedls is shown in control cells (top row), and SA treated cells (bottom row), in each panel of images. Images in each row, from left to right, show: i) the ARE binding protein detected via a Cy3 labeled 2° Ab in green, ii) Hedls detected via a Cy5 labeled 2° Ab in red, iii) merged Cy3 and Cy5 image, iv) FRETc signal in thermal pseudocolor scale. The images were captured at the level of the nucleus. A color matched signal intensity scale is indicated for each image. All scale bars are 10 μm in length. Panel A: HuR and Hedls. The Cy5 intensity in the Hedls and merged images (top row) was increased for easier detection of the cytoplasmic PB. The FRETc signal was set to an intensity of 20 to indicate the lack of a specific protein interaction as an intensity of < 20 is considered nonspecific. Panel B: KSRP and Hedls. The Cy5 intensity in the Hedls and merged images (top row) was increased for easier detection. The FRETc signal was set to an intensity of 20 to indicate the lack of a specific protein interaction. Panel C: TIA-1 and Hedls.