Fig 1: Co-immunoprecipitation of endogenous septins with Sept2-EGFP. (a) Western blot of septins coimmunoprecipitated from NRK49F-Sept2-EGFP cells using anti-GFP nanobody beads. (b) Silver-stained gel of immunoprecipitated septin complexes. E, elution; FT, unbound fraction (flow-through); S, supernatant incubated with anti-GFP nanobody beads; W1–W3, washing steps. Indicated septins were identified by western blotting and mass spectrometry analysis of excised bands (see Section 2)
Fig 2: Design and initial characterization of a genome-edited NRK49F-Sept2 EGFP cell-line. (a) Strategy for the integration of EGFP into the rat Sept2 locus. Exons shown in thick black. Recombination site used by the integration matrix represented by a gray box. Left and right TAL effector binding domains (BDs) framed. Sept2 exon given in the uppercase. The integration matrix contains left (LHA) and right (RHA) homology arms for homologous recombination. EGFP is inserted directly before and in frame with Sept2 start codon (ATG, green). (b) Genomic PCR on the Sept2 locus. Successful integration of EGFP into the Sept2 locus results in a longer PCR product. Outcome for the wild type locus and single- and double-allelic integration shown. (c) Western blot analysis on total cell extracts from wild type and genome-edited cells immunoblotted for Sept2. The same amount of protein was loaded into each lane. (d) Confocal microscopy image of live genome-edited NRK49F-Sept2-EGFP cells and fixed wild type NRK49F cells immunostained for Sept2 (inset). Scale bars are 10 µm. (e) Immunofluorescence micrograph of NRK49F-Sept2-EGFP cell-line showing EGFP fluorescence (green) and actin or tubulin staining (red). Sept2-EGFP decorates actin cables, but does not co-localize with tubulin in genome-edited NRK49F cells. Scale bars are 10 µm and 1 µm in the insets
Fig 3: Septin expression in homozygous genome-edited NRK49F-Sept2-EGFP cells. (a) Immunofluorescence micrographs showing septin immunofluorescence staining (left), Sept2-EGFP fluorescence (right) and merged images. Scale bars: 10 µm and 1 µm. (b) Western blot detection of septins in a total lysate from the genome-edited and wild type cell lines. (c) Real-time PCR analysis of cell lysate from wt and homozygous genome-edited cell lines. Shown is the detected mRNA level in homozygous genome-edited cell line relative to the wt expression level. Error bars are standard deviation
Fig 4: Cytokinesis and cell migration of the generated cells is not perturbed by the integration of EGFP into Sept2 locus. (a) The average percentage of binucleated cells in wild type and genome-edited cells. (b) Quantification of migration speeds of wild type and genome-edited cells in a wound-healing assay. (c) Kinetics of wound closure for wild type and genome-edited cells in a wound-healing assay
Fig 5: Comparison of Sept2 distribution during cell division in wild type and genome-edited cells. (a) Confocal images of wild type (left) and genome-edited (right) NRK49F fibroblast cells in different phases of the cell cycle. Sept2 antibody and Sept2-EGFP signal in green. Tubulin staining in red. (b) Individual frames from a live-cell time-lapse acquisition of dividing NRK49-Sept2-EGFP cells. Scale bars 10 µm
Supplier Page from Thermo Fisher Scientific for SEPT6 Antibody