Fig. 2

Jacob associates with the inner nuclear envelope via direct binding to LaminB1. a Schematic representation of the nuclear localization signal of Jacob and its conservation among species. b Jacob lacking the NLS does not associate with the nuclear envelope. Depicted are confocal images of a hippocampal primary neuron (17DIV) expressing ΔNLS-Jacob-GFP and co-labeled with anti-MAP2 and DAPI. c, d. Immunogold pre-embedding (c) and cryosection (d) labeling of Jacob in rat cortical pyramidal neurons. c Silver-enhanced gold particles of irregular shape were observed predominantly along the inner leaflet (arrowheads) of the nuclear envelope. d On thin Tokuyasu cryo-sections, Jacob immunosignal (10 nm gold particles) was observed at the inside of the nuclear envelope (arrowheads). Note the vesicular structure closely associated with the outer nuclear membrane (arrow). The boxed area is shown at increased magnification in the inset. Cy cytoplasm, Nu  nucleus. Scale bars are: 1 µm in (c); 500 nm in (d); 200 nm in inset in (d). e–g. STED imaging confirms a tide association of Jacob with laminB1 in hippocampal primary neurons as indicated with the line profile (g; line in f). Upper panel (e) represents Jacob and LaminB1 labeling with confocal resolution; lower panel represents STED images. Box in the merged image indicate the area selected for higher magnification image (f). Scale bar in (e) indicates 10 µm. Scale bar in (f) indicates 1 µm. h Co-Immunoprecipitation analysis revealed that Jacob and LaminB1 are part of one protein complex. i, j WT-Jacob-GFP is recruited to nuclear invaginations which result from tRFP-LaminB1 overexpression, indicated by the line profile. k Jacob directly binds LaminB1 as confirmed by a pull-down assay with recombinant proteins. Schematic represents the time-line of the experiment. An intein-tag was used for LaminB1 purification and was cleaved by addition of DTT. Anti-LaminB1 antibodies were used for detection of recombinant protein. l–n Jacob directly associate with LaminB1 in hippocampal primary neurons as confirmed by proximity ligation assay. l Schematic representation of the PLA assay between Jacob and LaminB1. NL nuclear lamina. m Confocal images of hippocampal primary neurons showing the signal of proximity ligation at the nuclear periphery. Right panel represents deconvolved confocal maximal projection images of nuclear PLA. Scale bar indicates 10 µm. n STED image of nuclear proximity ligation between Jacob and LaminB1. Scale bar indicates 1 µm