Fig 1: Functional interplay between EBLN1 and TPR.(A) Representative immunofluorescence images showing endogenous TPR staining in interphase U2OS cells transfected with the indicated siRNA. Additional examples are given in Supplementary Figure S1I. (B) Upper panel; representative immunofluorescence images of microtubule organisation (atubulin staining; green, ?tubulin; red and DAPI; blue) in interphase U2OS cells treated with either control or TPR siRNA as indicated. White arrows indicate the centrosomes (microtubule organising centres) that are shown enlarged in the inset images. Lower panel; quantification of organised microtubule (MT) arrays in interphase U2OS cells treated with either control or TPR siRNA. Data shown represents the mean from three independent experiments with associated SEMs (*p = 0.05 and **p = 0.01 compared to control siRNA cells). (C) Left panel; representative immunofluorescence images for centrosomes (?tubulin; red) in control and TPR siRNA treated U2OS cells. White arrows indicate the centrosomes shown in the enlarged images below. Right panel; quantification of centrosome splitting defects observed in cells treated with either non-targeting control or TPR siRNA. Data shown represents the mean from at least three independent experiments with associated SEMs (**p = 0.01 compared to control siRNA cells). (D) Left panel; representative immunofluorescent images of ?H2AX in control non-targeting and TPR siRNA treated U2OS cells, with graph on right showing average number of ?H2AX positive cells in each siRNA transfected population. Data shown represents the mean from at least three independent experiments with associated SEMs (*p = 0.05 and **p = 0.01 compared to control siRNA cells).
Fig 2: EBLN1 does not interact with the Cyclin B1-CDK1 complex, but does interact with TPR.(A) Immunoprecipitation of endogenous EBLN1 from HeLa cells probed with either EBLN1 or Cyclin B1 antibodies. Agarose beads incubated with cell extracts and IgG was used as a negative control for non-specific protein binding. Black arrow indicates FLAG-tagged EBLN1 band. (B) Immunoprecipitation (IP) of endogenous Cyclin B1 from asynchronous (upper panel) or mitotic (lower panel) FLAG-EBLN1 expressing HeLa cells probed with the indicated antibodies. The minus tetracycline (-Tet) samples (uninduced expression of FLAG-EBLN1) serve as negative controls for non-specific protein binding. (C) Immunoprecipitation of FLAG-EBLN1 from tetracycline-inducible HeLa cells probed with the indicated antibodies. Minus tetracycline samples serve as negative controls for non-specific protein binding. (D) Upper panel shows EBLN1 and FLAG western blots of eluates from FLAG-EBLN1 expressing tet-inducible HeLa cells. Lower panel shows a SYPRO Ruby stained polyacrylamide gel of FLAG eluates shown in the upper panel. Black arrow indicates FLAG-tagged EBLN1 band. (E) Table showing some of the most prevalent proteins co-immunoprecipitating with FLAG-EBLN1 as determined by proteomic analyses of the eluates shown in (E). The number of unique peptides for each protein is shown for both uninduced and induced (-Tet and + Tet) samples to highlight enrichment in FLAG-EBLN1 eluates (+Tet samples), along with the respective peptide coverage for each protein identified. (F) Indicated western blots on inputs (left panel), GFP immunoprecipitations and TPR immunoprecipitations (right panels) in stable tetracycline-inducible GFP-EBLN1 expressing HeLa cell lines. Arrows highlight GFP-EBLN1 band in each IP. Note that a longer exposure is shown for the TPR IPs compared with the GFP IPs. (G) TPR western blots of immunoprecipitated endogenous TPR from HeLa cells transfected with the indicated siRNA. Arrows highlight TPR isoforms (upper panel) and TPR-specific band in EBLN1 IPs (lower panel), which are reduced in cells transfected with TPR siRNA. Note that the TPR antibody is not capable of recognising endogenous TPR in the input lanes, only purified TPR in the IP lanes.
Fig 3: Export directionality of endogenous mRNAs and lncRNAs.a MKI67, TPR, NCOA3 mRNAs and TUG1 lncRNA double-tagged transcripts in their 5' and middle regions (5’—magenta, middle—cyan) together with POM121-Cer labeling (gray). Charts describing the percentage of transcripts exiting the pore in a 5’-manner (magenta) or middle-first (cyan). MKI67 n = 105, TPR n = 50, NCOA3 n = 53, TUG1 n = 30 transcripts. Dotted line—nuclear borders; cytoplasm above and nucleus below the line. Scale bar, 0.5 µm. b Double-tagged MKI67 transcripts (5’—magenta, middle—cyan) with endogenous Nup153-mNeonGreen labeling (green). Enlargements are shown in boxes. Scale bar, 8 µm. c Charts describing the percentage of transcripts exiting the pore in a 5’-manner (magenta) or middle-first (cyan) in cells expressing Nup153-mNeonGreen. MKI67 n = 121, TUG1 n = 50, NORAD n = 40 transcripts. Adjustments to individual color channels were made. Source data are provided as a Source Data file.
Supplier Page from Abcam for Anti-TPR antibody [EPR8982]