Journal: bioRxiv

Article Title: Distinct Basket Nucleoporins roles in Nuclear Pore Function and Gene Expression: Tpr is an integral component of the TREX-2 mRNA export pathway

doi: 10.1101/685263

Figure Lengend Snippet: a , Live imaging of Nup50, Nup153 and Tpr degradation. Chromatin is visualized by RCC1-IRFP fluorescent protein. Scale bar is 10 μ m. The green fluorescence intensity at the nuclear envelope corresponds to tagged nucleoporins in the absence or presence of auxin. The median degradation times for Nup50, Nup153 and Tpr were 60, 40, and 20 min, respectively. b , Growth rate of DLD-1 cells in the absence or presence of auxin. c, g, l , Protein regions of RanGAP1, NXF1, and GANP detected by respective antibodies. d, h, m Growth rates of AID-tagged RanGAP1, NXF1, or GANP1 cells upon auxin treatment for 1-3 days. Error bars are SD. p-value *** < 0.0005 (unpaired t-test). e, i, n , Genomic PCR demonstrating the integration of the AID degron sequence into the locus of rangap1, nxf1 , or ganp genes. f, j, o , The molecular weight shift and degradation of endogenous RanGAP1, NXF1, and GANP were analyzed by Western blot upon auxin treatment. Black and green arrows indicate the molecular weight of endogenous and tagged proteins.

Article Snippet: Specific primary antibodies against Nup50 (A301-782A), Nup153 (A301-789A), Tpr (A300-828A), GANP (A303-128A WB, A303-127A IF), NXF1 (A303-913A IF, A303-915A WB), Nup133 (A302-386A), Nup98 (sc-30112), HA (11867423001), FLAG M2 (F1804), Actin (13E5, 4970S), SON (GTX129778) were purchased from Bethyl Laboratories, Santa Cruz Biotechnology, Sigma-Aldrich, and GeneTex, respectively.

Techniques: Imaging, Fluorescence, Sequencing, Molecular Weight, Western Blot

Journal: bioRxiv

Article Title: Distinct Basket Nucleoporins roles in Nuclear Pore Function and Gene Expression: Tpr is an integral component of the TREX-2 mRNA export pathway

doi: 10.1101/685263

Figure Lengend Snippet: a, Localization of Nup98, Nup133, and RanBP2 nucleoporins on the NE in the absence or presence of Nup50 (4 h of auxin treatment). b-c , Localization of Nup153 ( b ) and Tpr ( c ) on the NE in the absence or presence of Nup50 (4 h of auxin treatment). Scale bar is 5 μ m. d, f Nup153-Nup50-Tpr interdependence in interphase (pre-existing NPC (d) and post-mitotic NPC (f) upon loss of Nup153, Tpr, and Nup50. e , Localization of mCherry-tagged Tpr in Nup153-depleted post-mitotic cells. Tpr does not mislocalize from the NE upon Nup153 loss in the preexisting NPC, but fails to be imported to the nucleus in post-mitotic Nup153-depleted cells. g , A summary table of Tpr, GANP, and NXF1 interdependence based on mass-spectrometry and immunofluorescence data. Red font indicates change after auxin treatment. h , A heat map of TMT-based mass-spectrometry of TREX2 complex and TREX-2 associated proteins abundance in NPC-enriched nuclear extracts upon loss of BSK-NUPs, GANP or NXF1.

Article Snippet: Specific primary antibodies against Nup50 (A301-782A), Nup153 (A301-789A), Tpr (A300-828A), GANP (A303-128A WB, A303-127A IF), NXF1 (A303-913A IF, A303-915A WB), Nup133 (A302-386A), Nup98 (sc-30112), HA (11867423001), FLAG M2 (F1804), Actin (13E5, 4970S), SON (GTX129778) were purchased from Bethyl Laboratories, Santa Cruz Biotechnology, Sigma-Aldrich, and GeneTex, respectively.

Techniques: Mass Spectrometry, Immunofluorescence

Journal: bioRxiv

Article Title: Distinct Basket Nucleoporins roles in Nuclear Pore Function and Gene Expression: Tpr is an integral component of the TREX-2 mRNA export pathway

doi: 10.1101/685263

Figure Lengend Snippet: Loss of Tpr leads to rapid changes in mRNA abundance. a, b , A scheme of the experiment and heatmaps of unsupervised k-means clustering of differentially expressed genes 2 h after auxin treatment of cells expressing corresponding AID-tagged proteins. DLD – parental DLD1 cells treated with auxin. c , Venn diagram representing the number of RNAs that showed significant change (both up- and down-regulation) upon Nup50, Nup153, Tpr, and RanGAP1 loss. Some RNAs altered after loss of multiple nucleoporins are not shown to avoid confusion. The corresponding RNAs in each category: Nup153-Nup50-Tpr (1 gene), Nup50-Tpr-RanGAP1 (2 genes), Nup153-Tpr-RanGAP1 (1 gene), Nup153-Nup50-Tpr-RanGAP1 (2 genes). d , Venn diagram representing the number of significantly regulated transcripts upon Tpr, NXF1, or GANP depletion (p-value = 2.3e-117, Hypergeometric distribution test). e , Top GO-terms (*Biological Process) of individual differentially expressed (DE) RNAs upon loss of indicated nucleoporins, NXF1, or GANP (DE ≥ 30 %, adj. p-value < 0.05, Wald test). f , Venn diagram representing overlaps between significantly up- or down-regulated transcripts upon Tpr, NXF1, or GANP loss. Note that most of the Tpr/NXF1/GANP-dependent RNAs are downregulated upon Tpr, NXF1, or GANP loss.

Article Snippet: Specific primary antibodies against Nup50 (A301-782A), Nup153 (A301-789A), Tpr (A300-828A), GANP (A303-128A WB, A303-127A IF), NXF1 (A303-913A IF, A303-915A WB), Nup133 (A302-386A), Nup98 (sc-30112), HA (11867423001), FLAG M2 (F1804), Actin (13E5, 4970S), SON (GTX129778) were purchased from Bethyl Laboratories, Santa Cruz Biotechnology, Sigma-Aldrich, and GeneTex, respectively.

Techniques: Expressing

Journal: bioRxiv

Article Title: Distinct Basket Nucleoporins roles in Nuclear Pore Function and Gene Expression: Tpr is an integral component of the TREX-2 mRNA export pathway

doi: 10.1101/685263

Figure Lengend Snippet: Tpr is required for GANP but not NXF1 tethering to the NE and poly(A) RNA nuclear export. a , The nuclear-cytoplasmic distribution of poly(A) RNA in AID-NGTpr targeted cells using oligo(dT)-Quasar 670 probe 8 h after Tpr degradation. Poly(A) RNA accumulates in the nuclear speckles of auxin-treated Tpr cells. Scale bar, 10 μ m. b-c , Tpr loss mislocalizes GANP from the NE. b , A heat map of TMT-based mass-spectrometry of NPC-enriched nuclear extracts shows changes in GANP, NXF1, and Tpr abundance 2 h after auxin treatment. c, GANP localization at the NE depends on Tpr, but not on Nup153 or Nup50. d , NXF1 localization is not affected upon Tpr, Nup153, or Nup50 loss. e, f , GANP and NXF1 localization at the NE are independent of each other. Correspondent AID-tagged cell lines were treated with auxin for 2 h. Scale bar is 5 μ m.

Article Snippet: Specific primary antibodies against Nup50 (A301-782A), Nup153 (A301-789A), Tpr (A300-828A), GANP (A303-128A WB, A303-127A IF), NXF1 (A303-913A IF, A303-915A WB), Nup133 (A302-386A), Nup98 (sc-30112), HA (11867423001), FLAG M2 (F1804), Actin (13E5, 4970S), SON (GTX129778) were purchased from Bethyl Laboratories, Santa Cruz Biotechnology, Sigma-Aldrich, and GeneTex, respectively.

Techniques: Mass Spectrometry

Journal: bioRxiv

Article Title: Distinct Basket Nucleoporins roles in Nuclear Pore Function and Gene Expression: Tpr is an integral component of the TREX-2 mRNA export pathway

doi: 10.1101/685263

Figure Lengend Snippet: Tpr regulates RNA localization and abundance of GANP-dependent transcripts. a, b , fjx1, c-fos , and actb RNA localization 2 h after Tpr, GANP, or NXF1 loss ( a ). Ratio of cytoplasmic vs. nuclear (Cyt/Nu) fjx1 and c-fos transcripts after Tpr or GANP loss ( b ). Asterisks indicate p-value ** < 0.01, *** < 0.001 (unpaired t-test). c , Schematic representation of Tpr’s role in gene expression through TREX-2-dependent mRNA export. Tpr anchors GANP to the NPC, and Tpr loss causes retention of GANP-dependent RNAs within the nucleus which affects their stability.

Article Snippet: Specific primary antibodies against Nup50 (A301-782A), Nup153 (A301-789A), Tpr (A300-828A), GANP (A303-128A WB, A303-127A IF), NXF1 (A303-913A IF, A303-915A WB), Nup133 (A302-386A), Nup98 (sc-30112), HA (11867423001), FLAG M2 (F1804), Actin (13E5, 4970S), SON (GTX129778) were purchased from Bethyl Laboratories, Santa Cruz Biotechnology, Sigma-Aldrich, and GeneTex, respectively.

Techniques: Gene Expression

Journal: bioRxiv

Article Title: Distinct Basket Nucleoporins roles in Nuclear Pore Function and Gene Expression: Tpr is an integral component of the TREX-2 mRNA export pathway

doi: 10.1101/685263

Figure Lengend Snippet: Stability of the assembled nuclear pore upon rapid loss of BSK-NUPs. a , A strategy of CRISPR/Cas9-based tagging of nucleoporin genes with AID sequence and degradation of the AID-fused proteins. b , Heat maps of differential abundance of Nup153, Nup50, and Tpr proteins in NPC-enriched nuclear extracts of indicated AID-tagged cells 2 h after auxin treatment using TMT-assisted and conventional (Label-free) mass-spectrometry. c , Localization of Nup98, Nup133, and RanBP2 in the absence or presence of Tpr and Nup153 (4 h of auxin treatment). d , Localization of BSK-NUPs in the absence or presence of Tpr and Nup153 (4 h of auxin treatment). Plots show line scans of relative fluorescence intensity (RFU) for BSK-NUPs and DNA across the nucleus. DNA was counterstained with DAPI. Note that Nup50 no longer localizes to the NE in the absence of Nup153. NE – nuclear envelope. Scale bar is 5 μ m.

Article Snippet: Specific primary antibodies against Nup50 (A301-782A), Nup153 (A301-789A), Tpr (A300-828A), GANP (A303-128A WB, A303-127A IF), NXF1 (A303-913A IF, A303-915A WB), Nup133 (A302-386A), Nup98 (sc-30112), HA (11867423001), FLAG M2 (F1804), Actin (13E5, 4970S), SON (GTX129778) were purchased from Bethyl Laboratories, Santa Cruz Biotechnology, Sigma-Aldrich, and GeneTex, respectively.

Techniques: CRISPR, Sequencing, Mass Spectrometry, Fluorescence

Journal: bioRxiv

Article Title: Distinct Basket Nucleoporins roles in Nuclear Pore Function and Gene Expression: Tpr is an integral component of the TREX-2 mRNA export pathway

doi: 10.1101/685263

Figure Lengend Snippet: a-b , A scheme of endogenous targeting, position of homologous arms, and primer sets for genotyping of C-(a) and N-(b) terminally targeted genes. F in -R in and F out -R out primer sets anneal inside and outside of the homologous arms, respectively. We biallelically tagged Nup50, Nup153 and Tpr with an AID and a N eon G reen (NG) fluorescent protein, using CRISPR/Cas9 to modify their endogenous loci in DLD1 cells. c , A scheme of TIR1 genomic integration into the rcc1 gene locus. d, h, l , Regions of Nup153, Nup50, and Tpr used for antibodies production (Bethyl Laboratories). e, i, m , Genomic PCR of homozygous clones demonstrating the integration of the NG-AID-P2A-Hygromycin sequence into genomic loci of nup50, nup153, and tpr genes. f, j, n , Degradation of Nup50 ( f ), Nup153 ( j ), and Tpr ( n ) after 1 h of auxin treatment. Black and green arrows indicate the molecular weight of unmodified and AID-NeonGreen (AID-NG) tagged nucleoporins. g, k, o , Growth rate of DLD-1 cells upon loss of Nup50, Nup153, and Tpr. Without auxin, AID-tagged cell lines were viable and did not show any obvious defects. Auxin addition led to rapid degradion of AID-tagged proteins and growth arrest of AID-Nup153 and AID-Tpr cells in the continuous presence of auxin. Error bars are SD. p-value *** < 0.0005 (unpaired t-test).

Article Snippet: Specific primary antibodies against Nup50 (A301-782A), Nup153 (A301-789A), Tpr (A300-828A), GANP (A303-128A WB, A303-127A IF), NXF1 (A303-913A IF, A303-915A WB), Nup133 (A302-386A), Nup98 (sc-30112), HA (11867423001), FLAG M2 (F1804), Actin (13E5, 4970S), SON (GTX129778) were purchased from Bethyl Laboratories, Santa Cruz Biotechnology, Sigma-Aldrich, and GeneTex, respectively.

Techniques: CRISPR, Clone Assay, Sequencing, Molecular Weight

Journal: bioRxiv

Article Title: Distinct Basket Nucleoporins roles in Nuclear Pore Function and Gene Expression: Tpr is an integral component of the TREX-2 mRNA export pathway

doi: 10.1101/685263

Figure Lengend Snippet: a , Live imaging of Nup50, Nup153 and Tpr degradation. Chromatin is visualized by RCC1-IRFP fluorescent protein. Scale bar is 10 μ m. The green fluorescence intensity at the nuclear envelope corresponds to tagged nucleoporins in the absence or presence of auxin. The median degradation times for Nup50, Nup153 and Tpr were 60, 40, and 20 min, respectively. b , Growth rate of DLD-1 cells in the absence or presence of auxin. c, g, l , Protein regions of RanGAP1, NXF1, and GANP detected by respective antibodies. d, h, m Growth rates of AID-tagged RanGAP1, NXF1, or GANP1 cells upon auxin treatment for 1-3 days. Error bars are SD. p-value *** < 0.0005 (unpaired t-test). e, i, n , Genomic PCR demonstrating the integration of the AID degron sequence into the locus of rangap1, nxf1 , or ganp genes. f, j, o , The molecular weight shift and degradation of endogenous RanGAP1, NXF1, and GANP were analyzed by Western blot upon auxin treatment. Black and green arrows indicate the molecular weight of endogenous and tagged proteins.

Article Snippet: Specific primary antibodies against Nup50 (A301-782A), Nup153 (A301-789A), Tpr (A300-828A), GANP (A303-128A WB, A303-127A IF), NXF1 (A303-913A IF, A303-915A WB), Nup133 (A302-386A), Nup98 (sc-30112), HA (11867423001), FLAG M2 (F1804), Actin (13E5, 4970S), SON (GTX129778) were purchased from Bethyl Laboratories, Santa Cruz Biotechnology, Sigma-Aldrich, and GeneTex, respectively.

Techniques: Imaging, Fluorescence, Sequencing, Molecular Weight, Western Blot

Journal: bioRxiv

Article Title: Distinct Basket Nucleoporins roles in Nuclear Pore Function and Gene Expression: Tpr is an integral component of the TREX-2 mRNA export pathway

doi: 10.1101/685263

Figure Lengend Snippet: a, Localization of Nup98, Nup133, and RanBP2 nucleoporins on the NE in the absence or presence of Nup50 (4 h of auxin treatment). b-c , Localization of Nup153 ( b ) and Tpr ( c ) on the NE in the absence or presence of Nup50 (4 h of auxin treatment). Scale bar is 5 μ m. d, f Nup153-Nup50-Tpr interdependence in interphase (pre-existing NPC (d) and post-mitotic NPC (f) upon loss of Nup153, Tpr, and Nup50. e , Localization of mCherry-tagged Tpr in Nup153-depleted post-mitotic cells. Tpr does not mislocalize from the NE upon Nup153 loss in the preexisting NPC, but fails to be imported to the nucleus in post-mitotic Nup153-depleted cells. g , A summary table of Tpr, GANP, and NXF1 interdependence based on mass-spectrometry and immunofluorescence data. Red font indicates change after auxin treatment. h , A heat map of TMT-based mass-spectrometry of TREX2 complex and TREX-2 associated proteins abundance in NPC-enriched nuclear extracts upon loss of BSK-NUPs, GANP or NXF1.

Article Snippet: Specific primary antibodies against Nup50 (A301-782A), Nup153 (A301-789A), Tpr (A300-828A), GANP (A303-128A WB, A303-127A IF), NXF1 (A303-913A IF, A303-915A WB), Nup133 (A302-386A), Nup98 (sc-30112), HA (11867423001), FLAG M2 (F1804), Actin (13E5, 4970S), SON (GTX129778) were purchased from Bethyl Laboratories, Santa Cruz Biotechnology, Sigma-Aldrich, and GeneTex, respectively.

Techniques: Mass Spectrometry, Immunofluorescence

Journal: bioRxiv

Article Title: Distinct Basket Nucleoporins roles in Nuclear Pore Function and Gene Expression: Tpr is an integral component of the TREX-2 mRNA export pathway

doi: 10.1101/685263

Figure Lengend Snippet: a, Co-localization of Nup50 and Mab414-positive nucleoporins in the absence or presence of Nup153 (4 h of auxin treatment, scale bar is 5 μ m). b , A scheme of mCherry integration into genomic loci of nup153, nup50, and tpr genes. c-h , mCherry-tagged Nup153, Nup50 or Tpr were integrated into corresponding AID-BSK-NUPs cell lines and were used to follow Nup153-Tpr-Nup50 interdependency. The representative images of the mCherry signal at 30 and 60 min after auxin addition are shown. Scale bar is 10 μ m.

Article Snippet: Specific primary antibodies against Nup50 (A301-782A), Nup153 (A301-789A), Tpr (A300-828A), GANP (A303-128A WB, A303-127A IF), NXF1 (A303-913A IF, A303-915A WB), Nup133 (A302-386A), Nup98 (sc-30112), HA (11867423001), FLAG M2 (F1804), Actin (13E5, 4970S), SON (GTX129778) were purchased from Bethyl Laboratories, Santa Cruz Biotechnology, Sigma-Aldrich, and GeneTex, respectively.

Techniques:

Journal: bioRxiv

Article Title: Distinct Basket Nucleoporins roles in Nuclear Pore Function and Gene Expression: Tpr is an integral component of the TREX-2 mRNA export pathway

doi: 10.1101/685263

Figure Lengend Snippet: a, c , Representative images of the import ( a ) export ( c ) assays. Scale bar, 10 μ m. In the import assay, a REV-GR-Turquiose2 substrate is localized in the cytoplasm. Upon treatment with dexamethasone, REV-GR-Turquiose2 becomes re-distributed to the nucleus. Import is measured by quantitation of Turquiose2 signal for 10 min immediately after dexamethasone addition. In the export assay, a mCherry-LOV2-BiLINuS2 substrate in loaded in the nucleus via blue light exposure. The mCherry-LOV2-BiLINuS2 shuttles back to the cytoplasm when blue light is switched off. Export is measured by quantitation of mCherry signal for 10 min immediately after removal of blue light. Import ( b ) and export ( d ) rates of model substrates in AID-tagged Nup50, Nup153, Tpr, and RanGAP1 cells in the presence (blue or red lines) or absence (black lines) of auxin. Error bars are MAD. e , A heat map of RNA Pol II Ser5P binding of AID-Tpr samples in the absence (0 hr) and presence (2hr) of auxin treatment. A region (± 2 kb) from transcription start site (TSS) and transcription end site (TES) is shown. Input samples did not show RNA Pol II Ser5P enrichment at the TSS or TES sites. f , Effect of BSK-NUPs and RanGAP1 depletion on the nuclear-cytoplasmic distribution of poly(A) RNA. Nup153, Tpr, Nup50, and RanGAP1 NG-AID targeted cells were analyzed using oligo(dT)-Quasar 670 probe. Cells were treated with 1 mM auxin for 8h. Representatives images from two independent experiments. Scale bar, 10 μ m. h , Poly(A) RNA export quantification in AID-tagged Nup50, Nup153, and Tpr cells in the presence or absence of auxin 8 h after auxin treatment. Error bars are SD. **< 0.005 for non-parametric t-test. i , c-fos and fjx1 RNA localization in DLD1-RanGAP1 -NG-AID cells in the absence and presence of auxin. c-fos and fjx1 RNA abundance is not changed upon RanGAP1 loss.

Article Snippet: Specific primary antibodies against Nup50 (A301-782A), Nup153 (A301-789A), Tpr (A300-828A), GANP (A303-128A WB, A303-127A IF), NXF1 (A303-913A IF, A303-915A WB), Nup133 (A302-386A), Nup98 (sc-30112), HA (11867423001), FLAG M2 (F1804), Actin (13E5, 4970S), SON (GTX129778) were purchased from Bethyl Laboratories, Santa Cruz Biotechnology, Sigma-Aldrich, and GeneTex, respectively.

Techniques: Quantitation Assay, Binding Assay

Journal: bioRxiv

Article Title: Distinct Basket Nucleoporins roles in Nuclear Pore Function and Gene Expression: Tpr is an integral component of the TREX-2 mRNA export pathway

doi: 10.1101/685263

Figure Lengend Snippet: Loss of Tpr leads to rapid changes in mRNA abundance. a, b , A scheme of the experiment and heatmaps of unsupervised k-means clustering of differentially expressed genes 2 h after auxin treatment of cells expressing corresponding AID-tagged proteins. DLD – parental DLD1 cells treated with auxin. c , Venn diagram representing the number of RNAs that showed significant change (both up- and down-regulation) upon Nup50, Nup153, Tpr, and RanGAP1 loss. Some RNAs altered after loss of multiple nucleoporins are not shown to avoid confusion. The corresponding RNAs in each category: Nup153-Nup50-Tpr (1 gene), Nup50-Tpr-RanGAP1 (2 genes), Nup153-Tpr-RanGAP1 (1 gene), Nup153-Nup50-Tpr-RanGAP1 (2 genes). d , Venn diagram representing the number of significantly regulated transcripts upon Tpr, NXF1, or GANP depletion (p-value = 2.3e-117, Hypergeometric distribution test). e , Top GO-terms (*Biological Process) of individual differentially expressed (DE) RNAs upon loss of indicated nucleoporins, NXF1, or GANP (DE ≥ 30 %, adj. p-value < 0.05, Wald test). f , Venn diagram representing overlaps between significantly up- or down-regulated transcripts upon Tpr, NXF1, or GANP loss. Note that most of the Tpr/NXF1/GANP-dependent RNAs are downregulated upon Tpr, NXF1, or GANP loss.

Article Snippet: Specific primary antibodies against Nup50 (A301-782A), Nup153 (A301-789A), Tpr (A300-828A), GANP (A303-128A WB, A303-127A IF), NXF1 (A303-913A IF, A303-915A WB), Nup133 (A302-386A), Nup98 (sc-30112), HA (11867423001), FLAG M2 (F1804), Actin (13E5, 4970S), SON (GTX129778) were purchased from Bethyl Laboratories, Santa Cruz Biotechnology, Sigma-Aldrich, and GeneTex, respectively.

Techniques: Expressing

Journal: bioRxiv

Article Title: Distinct Basket Nucleoporins roles in Nuclear Pore Function and Gene Expression: Tpr is an integral component of the TREX-2 mRNA export pathway

doi: 10.1101/685263

Figure Lengend Snippet: Tpr is required for GANP but not NXF1 tethering to the NE and poly(A) RNA nuclear export. a , The nuclear-cytoplasmic distribution of poly(A) RNA in AID-NGTpr targeted cells using oligo(dT)-Quasar 670 probe 8 h after Tpr degradation. Poly(A) RNA accumulates in the nuclear speckles of auxin-treated Tpr cells. Scale bar, 10 μ m. b-c , Tpr loss mislocalizes GANP from the NE. b , A heat map of TMT-based mass-spectrometry of NPC-enriched nuclear extracts shows changes in GANP, NXF1, and Tpr abundance 2 h after auxin treatment. c, GANP localization at the NE depends on Tpr, but not on Nup153 or Nup50. d , NXF1 localization is not affected upon Tpr, Nup153, or Nup50 loss. e, f , GANP and NXF1 localization at the NE are independent of each other. Correspondent AID-tagged cell lines were treated with auxin for 2 h. Scale bar is 5 μ m.

Article Snippet: Specific primary antibodies against Nup50 (A301-782A), Nup153 (A301-789A), Tpr (A300-828A), GANP (A303-128A WB, A303-127A IF), NXF1 (A303-913A IF, A303-915A WB), Nup133 (A302-386A), Nup98 (sc-30112), HA (11867423001), FLAG M2 (F1804), Actin (13E5, 4970S), SON (GTX129778) were purchased from Bethyl Laboratories, Santa Cruz Biotechnology, Sigma-Aldrich, and GeneTex, respectively.

Techniques: Mass Spectrometry

Journal: bioRxiv

Article Title: Distinct Basket Nucleoporins roles in Nuclear Pore Function and Gene Expression: Tpr is an integral component of the TREX-2 mRNA export pathway

doi: 10.1101/685263

Figure Lengend Snippet: Stability of the assembled nuclear pore upon rapid loss of BSK-NUPs. a , A strategy of CRISPR/Cas9-based tagging of nucleoporin genes with AID sequence and degradation of the AID-fused proteins. b , Heat maps of differential abundance of Nup153, Nup50, and Tpr proteins in NPC-enriched nuclear extracts of indicated AID-tagged cells 2 h after auxin treatment using TMT-assisted and conventional (Label-free) mass-spectrometry. c , Localization of Nup98, Nup133, and RanBP2 in the absence or presence of Tpr and Nup153 (4 h of auxin treatment). d , Localization of BSK-NUPs in the absence or presence of Tpr and Nup153 (4 h of auxin treatment). Plots show line scans of relative fluorescence intensity (RFU) for BSK-NUPs and DNA across the nucleus. DNA was counterstained with DAPI. Note that Nup50 no longer localizes to the NE in the absence of Nup153. NE – nuclear envelope. Scale bar is 5 μ m.

Article Snippet: Specific primary antibodies against Nup50 (A301-782A), Nup153 (A301-789A), Tpr (A300-828A), GANP (A303-128A WB, A303-127A IF), NXF1 (A303-913A IF, A303-915A WB), Nup133 (A302-386A), Nup98 (sc-30112), HA (11867423001), FLAG M2 (F1804), Actin (13E5, 4970S), SON (GTX129778) were purchased from Bethyl Laboratories, Santa Cruz Biotechnology, Sigma-Aldrich, and GeneTex, respectively.

Techniques: CRISPR, Sequencing, Mass Spectrometry, Fluorescence

Journal: bioRxiv

Article Title: Distinct Basket Nucleoporins roles in Nuclear Pore Function and Gene Expression: Tpr is an integral component of the TREX-2 mRNA export pathway

doi: 10.1101/685263

Figure Lengend Snippet: a, Localization of Nup98, Nup133, and RanBP2 nucleoporins on the NE in the absence or presence of Nup50 (4 h of auxin treatment). b-c , Localization of Nup153 ( b ) and Tpr ( c ) on the NE in the absence or presence of Nup50 (4 h of auxin treatment). Scale bar is 5 μ m. d, f Nup153-Nup50-Tpr interdependence in interphase (pre-existing NPC (d) and post-mitotic NPC (f) upon loss of Nup153, Tpr, and Nup50. e , Localization of mCherry-tagged Tpr in Nup153-depleted post-mitotic cells. Tpr does not mislocalize from the NE upon Nup153 loss in the preexisting NPC, but fails to be imported to the nucleus in post-mitotic Nup153-depleted cells. g , A summary table of Tpr, GANP, and NXF1 interdependence based on mass-spectrometry and immunofluorescence data. Red font indicates change after auxin treatment. h , A heat map of TMT-based mass-spectrometry of TREX2 complex and TREX-2 associated proteins abundance in NPC-enriched nuclear extracts upon loss of BSK-NUPs, GANP or NXF1.

Article Snippet: Specific primary antibodies against Nup50 (A301-782A), Nup153 (A301-789A), Tpr (A300-828A), GANP (A303-128A WB, A303-127A IF), NXF1 (A303-913A IF, A303-915A WB), Nup133 (A302-386A), Nup98 (sc-30112), HA (11867423001), FLAG M2 (F1804), Actin (13E5, 4970S), SON (GTX129778) were purchased from Bethyl Laboratories, Santa Cruz Biotechnology, Sigma-Aldrich, and GeneTex, respectively.

Techniques: Mass Spectrometry, Immunofluorescence

Journal: bioRxiv

Article Title: Distinct Basket Nucleoporins roles in Nuclear Pore Function and Gene Expression: Tpr is an integral component of the TREX-2 mRNA export pathway

doi: 10.1101/685263

Figure Lengend Snippet: Stability of the assembled nuclear pore upon rapid loss of BSK-NUPs. a , A strategy of CRISPR/Cas9-based tagging of nucleoporin genes with AID sequence and degradation of the AID-fused proteins. b , Heat maps of differential abundance of Nup153, Nup50, and Tpr proteins in NPC-enriched nuclear extracts of indicated AID-tagged cells 2 h after auxin treatment using TMT-assisted and conventional (Label-free) mass-spectrometry. c , Localization of Nup98, Nup133, and RanBP2 in the absence or presence of Tpr and Nup153 (4 h of auxin treatment). d , Localization of BSK-NUPs in the absence or presence of Tpr and Nup153 (4 h of auxin treatment). Plots show line scans of relative fluorescence intensity (RFU) for BSK-NUPs and DNA across the nucleus. DNA was counterstained with DAPI. Note that Nup50 no longer localizes to the NE in the absence of Nup153. NE – nuclear envelope. Scale bar is 5 μ m.

Article Snippet: Specific primary antibodies against Nup50 (A301-782A), Nup153 (A301-789A), Tpr (A300-828A), GANP (A303-128A WB, A303-127A IF), NXF1 (A303-913A IF, A303-915A WB), Nup133 (A302-386A), Nup98 (sc-30112), HA (11867423001), FLAG M2 (F1804), Actin (13E5, 4970S), SON (GTX129778) were purchased from Bethyl Laboratories, Santa Cruz Biotechnology, Sigma-Aldrich, and GeneTex, respectively.

Techniques: CRISPR, Sequencing, Mass Spectrometry, Fluorescence

Journal: bioRxiv

Article Title: Distinct Basket Nucleoporins roles in Nuclear Pore Function and Gene Expression: Tpr is an integral component of the TREX-2 mRNA export pathway

doi: 10.1101/685263

Figure Lengend Snippet: a, Localization of Nup98, Nup133, and RanBP2 nucleoporins on the NE in the absence or presence of Nup50 (4 h of auxin treatment). b-c , Localization of Nup153 ( b ) and Tpr ( c ) on the NE in the absence or presence of Nup50 (4 h of auxin treatment). Scale bar is 5 μ m. d, f Nup153-Nup50-Tpr interdependence in interphase (pre-existing NPC (d) and post-mitotic NPC (f) upon loss of Nup153, Tpr, and Nup50. e , Localization of mCherry-tagged Tpr in Nup153-depleted post-mitotic cells. Tpr does not mislocalize from the NE upon Nup153 loss in the preexisting NPC, but fails to be imported to the nucleus in post-mitotic Nup153-depleted cells. g , A summary table of Tpr, GANP, and NXF1 interdependence based on mass-spectrometry and immunofluorescence data. Red font indicates change after auxin treatment. h , A heat map of TMT-based mass-spectrometry of TREX2 complex and TREX-2 associated proteins abundance in NPC-enriched nuclear extracts upon loss of BSK-NUPs, GANP or NXF1.

Article Snippet: Specific primary antibodies against Nup50 (A301-782A), Nup153 (A301-789A), Tpr (A300-828A), GANP (A303-128A WB, A303-127A IF), NXF1 (A303-913A IF, A303-915A WB), Nup133 (A302-386A), Nup98 (sc-30112), HA (11867423001), FLAG M2 (F1804), Actin (13E5, 4970S), SON (GTX129778) were purchased from Bethyl Laboratories, Santa Cruz Biotechnology, Sigma-Aldrich, and GeneTex, respectively.

Techniques: Mass Spectrometry, Immunofluorescence