Journal: Nature Communications

Article Title: Ndfip-mediated degradation of Jak1 tunes cytokine signalling to limit expansion of CD4+ effector T cells

doi: 10.1038/ncomms11226

Figure Lengend Snippet: ( a ) Immunoblot of Itch and Nedd4-2 isolated from activated WT or Ndfip1/Ndfip2 DKO CD4+ T-cell lysates by GST pulldown of Ndfip1 and Ndfip2 cytosolic domain GST fusion proteins. Coomassie stain for the GST fusion proteins revealed full-length products for each construct as well as a GST cleavage product. ( b ) Itch activity, in the absence or presence of Ndfip1 or Ndfip2, was analysed via TR-FRET polyubiquitylation assay. ( c ) E2/E3 transthiolation assay was used to test whether Ndfip2 promotes ubiquitin ‘charging' of Itch. Biotinylated ubiquitin non-covalently bound to Itch was analysed by western blot using fluorescent streptavidin (top); total protein was visualized by Coomassie stain (bottom). ( d , e ) As in b , human Nedd4L activity was assessed alone or in the presence of Ndfip1 or Ndfip2 by TR-FRET. Data for each panel is representative of a minimum of three independent experiments.

Article Snippet: Samples were rotated at 4 ° with 50 μg biotinylated pan-TUBE (UM301; Lifesensors) for 2 h. A volume of 250 μl per sample Dynabeads MyOne Streptavidin c1 (Invitrogen) was washed and added to samples, which were rotated for an additional 2 h at 4 °.

Techniques: Western Blot, Isolation, Staining, Construct, Activity Assay, Ubiquitin Proteomics