Journal: Nature Communications

Article Title: Assay to visualize specific protein oxidation reveals spatio-temporal regulation of SHP2

doi: 10.1038/s41467-017-00503-w

Figure Lengend Snippet: Spatio-temporal dynamics of SHP2 oxidation. a Serum-starved Swiss 3T3 cells were stimulated with PDGF-BB (50 ng ml −1 ) for the indicated times, and subjected to dimedone-PLA. Representative images are shown for each condition from one of two independent experiments. The graph shows the average number of PLA signals per cell ( n = 6 images for each condition, 5–20 cells in an image), relative to unstimulated control cells (normalized to 1). Scale bar : 50 μm. b Serum-starved Swiss 3T3 cells were stimulated with PDGF-BB (50 ng ml −1 ) for the indicated times. Dimedone-PLA ( magenta ) and co-staining with the indicated markers ( green ) are shown. Nuclei were stained with DAPI ( blue ). Representative images are shown for each condition from one of three independent experiments. Higher magnification images of the boxed regions are shown. Median distances of centers of mass ( CM ) between punctate signals of ox-SHP2 and the nearest indicated marker signal in cells were obtained by object-based image analysis. The box-whisker plots show the median inter-object distances at the indicated times after stimulation ( n = 50 cells, each time point). Boxes indicate the 25th–75th percentile; whiskers represent the 5th–95th percentile. *** P < 0.0001, ANOVA with Bonferroni/Dunn’s post-hoc test. Scale bar : 10 μm. c Serum-starved Swiss 3T3 cells were stimulated with PDGF-BB (50 ng ml −1 ) for 10 min, and subjected to dimedone-PLA ( magenta ) and co-staining with the indicated antibodies ( green ). Representative semi-super resolution microscopic images (AiryScan) and higher magnification images of the boxed region from one of two independent experiments are shown. Scale bars : 5 μm. d Swiss 3T3 cells expressing EGFP, EGFP-fused wild type dynamin2 (WT Dynamin2) or dominant-negative dynamin2 (dynamin2 K44A ) were serum-starved and stimulated with PDGF-BB (50 ng ml −1 ) or H 2 O 2 (1 mM) for 10 min, and subjected to dimedone-PLA. Representative images of co-staining of dimedone-PLA ( gray ) and EGFP ( green ) are shown for each condition from one of two independent experiments. The graph represents the average number of PLA signals per cell ( n = 15 cells), relative to unstimulated control cells (normalized to 1). *** P < 0.0001, ns not significant, ANOVA with Bonferroni/Dunn’s post-hoc test. Scale bar : 50 μm. Error bars represent SD

Article Snippet: Expression vectors for EGFP-fused RAB5A, RAB7A, RAB9A, and RAB11A and pEGFP-WT Dynamin2 and Dynamin2 K44A were purchased from Addgene.

Techniques: Control, Staining, Marker, Whisker Assay, Expressing, Dominant Negative Mutation