Figure 6

Localization and FRET interactions of endogenous ARE binding proteins upon MAPK activation: Movement to P bodies? ARE-BPs were detected via immunocytochemistry. Cells were treated with 75 nM anisomycin for 24 hours. PBs were defined by presence of Hedls. The localization of each ARE-BP and Hedls is shown in control cells (top row), and anisomycin treated cells (bottom row), in each panel of images. Images in each row, from left to right, show the localization of a protein detected via: i) a Cy3 labeled 2° Ab in green, ii) a Cy5 or AF647 labeled 2° Ab in red, iii) merged Cy3-Cy5 image, iv) FRETc signal in a thermal pseudocolor scale. A color matched signal intensity scale is indicated for each image. All scale bars are 10 μm in length. Cell outline as shown. Panel A: HuR (Cy3) and Hedls (Cy5). The FRETc signal was set to an intensity of 20 to indicate the lack of a specific protein interaction. A FRETc signal intensity less than 20 was considered nonspecific. The Cy5 signal intensity was increased in all images of Hedls for better visualization. Panel B: KSRP (Cy3) and Hedls (Cy5). The FRETc signal was set to an intensity of 20 to indicate the lack of a specific protein interaction. The Hedls Cy5 signal intensity was increased in all images for ease of PB detection. Panel C: Hedls (Cy3) and TIA-1 (AF647).