Fig. 7: Kapβ2W460A:W730A is more efficient in preventing poly(GR) recruitment into stress granules.
A Representative images showing recruitment of TAMRA-(GR)20, in the presence of WT Kapβ2 or Kapβ2W460A:W730A, to G3BP1-positive stress granules in semi-permeabilized HeLa cells that were treated with sodium arsenite. Scale bar as indicated in the figure. B Quantification of the mean fluorescence intensity of TAMRA-(GR)20 in stress granules in the presence of WT Kapβ2 or Kapβ2W460A:W730A. The number of stress granules (n) quantified in each condition is indicated in the figure. Unpaired Student’s t-tests were used to compare different conditions; ****p ≤ 0.0001. C Co-immunoprecipitation (Co-IP) of (GR)50-GFP or (GA)50-GFP with V5-tagged WT Kapβ2 or Kapβ2W460A:W730A, from lysate of primary cortical neurons transduced with hsyn::(GR)50-GFP or hsyn::(GA)50-GFP, and hsyn::WT Kapβ2 or hsyn::Kapβ2W460A:W730A. Antibodies used for Co-IP and western blots are indicated. The intensity of the V5 and GFP bands was quantified, and the ratio of the IP band intensity to the input band intensity (normalized to vinculin) is shown in the bottom panel. D Representative confocal images of primary cortical neurons transduced with hsyn::(GR)50-GFP alone, or hsyn::(GR)50-GFP with hsyn::WT Kapβ2 or hsyn::Kapβ2W460A:W730A and treated with 0.5 mM sodium arsenite for 30 minutes. GFP signal is shown in green, TIAR signal is shown in Red, and nuclei are shown in blue. Scale bar = 10 µm. Shown on the right are the intensity profiles along the indicated lines across the stress granule. E Quantification of the confocal images collected in (D) showing relative fluorescence intensity of (GR)50-GFP in stress granules marked by TIAR. n = 3, m = 10. Parametric, unpaired Student’s t-tests were used to compare different conditions.
