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Fig. 3 | Molecular Brain

Fig. 3

From: The nuclear lamina is a hub for the nuclear function of Jacob

Fig. 3

Jacob contains a nuclear export signal that is responsible for its association with the nuclear lamina and Jacob is actively exported from the nucleus via a CRM1- and RanGTP-dependent mechanism. a The primary structure of Jacob contains a Class1a CRM1-dependent NES that is conserved across species. A point mutation (L476A) was introduced in the Jacob sequence (indicated by asterisk). b A point mutation within the Jacob NES (L476A) altered the subcellular localization of the protein. Depicted are confocal images of a hippocampal neuron (DIV16/18) expressing mNES-Jacob-GFP. The nuclear localization of the mutant protein is altered with no accumulation at the nuclear envelope. Scale bar is 20 μm. c, d WT-Jacob but not the NES mutant directly associates with CRM1. c Pull-down assay performed with bacterially produced recombinant MBP-45-532-Jacob and HEK293T cell extract expressing CRM1. d Recombinant NES-Jacob mutant does not bind to recombinant CRM1 as confirmed by a pull-down assay. Schematic represents the timeline of the experiment. eh Jacob ectopically expressed in HEK293 cells shows association with the nuclear envelope that is clearly reduced in the presence of GTP-locked form of Ran pmCherry-RanQ69L, but not in the presence of RanGDP-locked mutant (T24N). Scale bars are 15 μm. I. WT-Jacob-GFP associates with the nuclear rim and this association is abolished upon the treatment with LMB (0.1 nM). The schematic drawing indicates the timeline of the time-lapse experiment. Scale bar indicates 10 μm. j, k Confocal images of hippocampal primary neurons (DIV16/18) treated with 0.1 nM LMB showing enhanced nuclear IR of endogenous Jacob. Images represented in color code look-up-table (Jacob LUT) show an uneven nuclear distribution. Cultures were fixed 30 min after treatment. The region of interest for quantification was identified based on a DAPI staining. (***) indicate p < 0.001 (ANOVA, Bonferroni). Scale bar indicates 10 μm

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