Extended Data Fig. 5: The interaction of DNAJB6 with hNup153FG depends on the FG-motifs in hNup153FG. | Nature Cell Biology

Extended Data Fig. 5: The interaction of DNAJB6 with hNup153FG depends on the FG-motifs in hNup153FG.

From: The chaperone DNAJB6 surveils FG-nucleoporins and is required for interphase nuclear pore complex biogenesis

Extended Data Fig. 5

a, Schematic overview of the BioID2-DNAJB6b fusion protein and how samples were obtained. Fluorescent image shows HEK293T cells expressing BioID2-DNAJB6b, treated for 24 h with 50 μM biotin and stained with Alexa Fluor 488-conjugated streptavidin, biotinylated substrates are visualized in foci at the NE. b, Streptavidin blot belonging Fig. 4a where HEK293T cells expressing V5-DNAJB6b or BioID2-DNAJB6b are treated for 24 h with 50 μM biotin. c, Proximity ligation assay (PLA) performed with antibodies against FG-Nups (mAb414) or DNAJB6 alone, or only both secondary antibodies as negative technical controls for PLA. d, SDS-PAGE showing increase in HSPA6 levels after heat shock (43 °C for 30 minutes) but not in DNAJB6 KO nor after overexpression of hNup153FG. e, GFP-Nup153 full length localizes predominantly to the NE in HEK293T cells. Due to overexpression, there is also small clustering at the NE, but no aggregates can be observed. f, GFP-Nup153AG overexpression in HEK293T cells is homogeneously distributed in the cytoplasm. g, HEK293T cells overexpressing GFP-Nup153FG and mCherry-DNAJB6b show colocalization in spherical accumulations (arrowheads). h, Filter-trap assay of HEK293T cells with GFP-Nup153AG or GFP-Nup153FG overexpression. Three serial 5-fold dilutions were loaded onto cellulose-acetate membranes and probed with anti-GFP antibodies to detect aggregation of the GFP-Nup153FG or GFP-Nup153AG fragment without or with co-overexpression of V5-DNAJB6b. i, HEK293T cells overexpressing V5-DNAJB6b or BioID2-DNAJB6b, either with GFP-Nup153FG or GFP-Nup153AG, are treated for 24 h with 50 μM biotin. Biotinylated close proximity partners of DNAJB6b are pulled down by biotin affinity immunoprecipitation with streptavidin beads, run on SDS-PAGE. GFP and DNAJB6 are detected with specific antibodies. Scale bars on all fluorescent images represent 10 μm. Source numerical data and unprocessed blots are available in source data.

Source data

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