Journal: Biochemistry and Biophysics Reports

Article Title: Acetylation of the nuclear localization signal in Ku70 diminishes the interaction with importin-α

doi: 10.1016/j.bbrep.2022.101418

Figure Lengend Snippet: Effect of substitution of lysine residues in the Ku70 NLS with acetyl–lysine on binding to Impα. (A) The effect of amino acid substitutions in the Ku70 NLS on Impα binding. The binding activity of Ku70 NLS and its mutant peptides to Impα in the presence of Impβ was analyzed by probing immunoblots with antibodies against Impα. (B) Quantification of the pull-down assays presented in panel (A) . Measurements of the blot band intensity were performed using ImageJ 1.52a. Each graph represents the relative intensity with Ku70 NLS WT defined as 100%. The error bars indicate the standard deviation from three independent experiments. *p < 0.05 significant differences from the case when the expected value was defined as 100.

Article Snippet: A binding assay was performed with 50 μl NLS-immobilized sepharose, 1 mg/ml bovine serum albumin, 0.1 μg recombinant human Impα2 (NBP1-78888; Novus Biologicals, Centennial, CO, USA), and 0.1 μg recombinant human Impβ (NBP1-78815; Novus Biologicals) in 0.5 ml transport buffer by incubation for 2 h at 4 °C with gentle rotation.

Techniques: Binding Assay, Activity Assay, Mutagenesis, Western Blot, Standard Deviation

Journal: PLoS ONE

Article Title: Ciliary Entry of the Hedgehog Transcriptional Activator Gli2 Is Mediated by the Nuclear Import Machinery but Differs from Nuclear Transport in Being Imp-α/β1-Independent

doi: 10.1371/journal.pone.0162033

Figure Lengend Snippet: (A) Two segments of a T-Coffee alignment of four Gli-like sequences: GLI2_ Mm ( Mus musculus ), GLI2_ H s, GLI1_ Hs , GLI3_ Hs ( Homo sapiens ) and Ci_ Dm ( Drosophila melanogaster ). Residue similarity is colour coded according to the Risler substitution matrix, using ESPript . Dots mark 10-residue intervals of the top sequence. Black bars indicate the two bipartite cNLS (cNLS-1 and cNLS-2) predicted by cNLS mapper for GLI2_ Mm . (B) GFP-Gli2-NIH/3T3 cells were treated with Importazol (IPZ) in order to inhibit Imp-β1-mediated nuclear transport. DMSO was used as control. Hh signalling was activated for 90 min using SAG and non-stimulated cells were treated with DMSO. Cells were stained for cilium (anti-Ac.Tub, red), GFP-Gli2 (anti-GFP, green) and nucleus (TOPRO, blue). The same pictures are also shown with the channel corresponding to GFP-Gli2 in grey scale to help the visualization. Scale bar: 10 μm. (C) Quantification of nuclear Gli2 was performed as explained in Materials and Methods. The mean nuclear fluorescence was normalised against the mean total GFP fluorescence of the cell, so as to correct for variations in Gli2 expression among different cells. Results are representative of three experiments and at least 60 cells were analysed for each condition. ** p<0.001, *** p<0.0001 (Kruskal-Wallis test). (D) Activation of a luciferase-based Hh reporter gene in NIH/3T3 cells stimulated with SAG (or DMSO as control) in the presence of IPZ or DMSO. As explained in Materials and Methods, RLU values from SAG treated cells are normalised against RLU of DMSO treated ones and expressed as mean ± s.d. from triplicates from two independent experiments. * p< 0.01 (Mann-Whitney test). (E) Western blot (WB) showing GFP-Gli2 levels in control and IPZ-treated cells. GFP-Gli2 was detected using an anti-GFP antibody. (F-G) WB detecting Imp-β1 (F) and Imp-α1 (G) after precipitating GFP-Gli2 with GFP-Trap (left) from HEK293FT transfected with pEGFP-Gli2 (left panels). Membranes were cut at different levels so as to detect in the same samples the precipitated GFP-Gli2 and GFP (left panels). The levels of GFP-Gli2, Imp-β1, Imp-α and GFP in the lysates used for immunoprecipitation were assessed by WB and are shown in the right panels. The band corresponding to GFP looks distorted because the protein migrates with the dye front.

Article Snippet: The primary antibodies used were anti-Gli2 (H-300 rabbit polyclonal, Santa Cruz Biotechnology, 1:500, or goat polyclonal, R&D Systems (AF3635), 1:1000, overnight at 4°C), anti-GFP (rabbit polyclonal, Life or Cell Signaling Technology, or rat polyclonal, ChromoTek, 1:1000, 2 h at room temperature), anti-Imp-α1(#MAB6207) and Imp-β1 (#MAB8209) (mouse monoclonals, R&D Systems, 1:7000 and 1:1000 respectively, overnight at 4°C), anti-Imp-β2 (mouse monoclonal, Abcam #ab10303, 1:1000, overnight at 4°C) and anti-α tubulin (mouse monoclonal, Sigma, 1:2000, 1 h at room temperature).

Techniques: Residue, Sequencing, Control, Staining, Fluorescence, Expressing, Activation Assay, Luciferase, MANN-WHITNEY, Western Blot, Transfection, Immunoprecipitation

Journal: PLoS ONE

Article Title: Ciliary Entry of the Hedgehog Transcriptional Activator Gli2 Is Mediated by the Nuclear Import Machinery but Differs from Nuclear Transport in Being Imp-α/β1-Independent

doi: 10.1371/journal.pone.0162033

Figure Lengend Snippet: (A) Sequences in Gli2_ Mm predicted as cNLSs by cNLS-Mapper using a cut-off of 5 with the corresponding scores (out of 10). Residues that were mutated for alanines in the mutNLS constructs are in bold. The mutated sequences are not predicted (n.p.) as cNLS. (B) Transduced NIH/3T3 cells expressing GFP-Gli2, wt or cNLS mutants (mutNLS-1, mutNLS-2 or mutNLS-1+2), were treated with SAG for 90 min or DMSO as control. Cells were stained for cilium (anti-Ac.Tub, red), GFP-Gli2 (anti-GFP, green) and nucleus (DAPI, blue). The same pictures are also shown with the channel corresponding to GFP-Gli2 in grey scale to help the visualization. Scale bar: 10 μm. (C) Quantification of nuclear Gli2 was performed as explained in . Black, solid line indicates comparison between control and Hh activated conditions for wtGli2, dotted line indicates comparison between wtGli2 under basal condition and mutNLS-1 or mutNLS-1+2 under basal or activated conditions and black, discontinued line indicates comparison between activated conditions for wtGl2 and mutNLS-2. *p<0.01, *** p<0.0001 (Kruskal-Wallis test). (B) and (C) are representative of four independent experiments and at least 70 cells were analysed for each condition. (D) WB detecting Imp-α1 after precipitating GFP-Gli2 or GFP-Gli2-mutNLS-1+2 with GFP-Trap from NIH/3T3 Flp-In expressing at endogenous levels the constructs mentioned above. Membranes were cut at different levels so as to detect in the same samples the precipitated GFP-Gli2 (using an anti-Gli2 antibody) and GFP (left panels).

Article Snippet: The primary antibodies used were anti-Gli2 (H-300 rabbit polyclonal, Santa Cruz Biotechnology, 1:500, or goat polyclonal, R&D Systems (AF3635), 1:1000, overnight at 4°C), anti-GFP (rabbit polyclonal, Life or Cell Signaling Technology, or rat polyclonal, ChromoTek, 1:1000, 2 h at room temperature), anti-Imp-α1(#MAB6207) and Imp-β1 (#MAB8209) (mouse monoclonals, R&D Systems, 1:7000 and 1:1000 respectively, overnight at 4°C), anti-Imp-β2 (mouse monoclonal, Abcam #ab10303, 1:1000, overnight at 4°C) and anti-α tubulin (mouse monoclonal, Sigma, 1:2000, 1 h at room temperature).

Techniques: Construct, Expressing, Control, Staining, Comparison

Journal: PLoS ONE

Article Title: Ciliary Entry of the Hedgehog Transcriptional Activator Gli2 Is Mediated by the Nuclear Import Machinery but Differs from Nuclear Transport in Being Imp-α/β1-Independent

doi: 10.1371/journal.pone.0162033

Figure Lengend Snippet: (A) NIH/3T3 cells transduced for expressing GFP-Gli2, wt or mutants in cNLS (mutNLS-1, mutNLS-2 or mutNLS-1+2) were treated with SAG for 90 min or DMSO as control. Cells were stained for cilium (anti-Ac.Tub, red) and GFP-Gli2 (anti-GFP, green). Images show representative cilia from each condition. Scale bar: 1 μm. (B) Quantification of Gli2 ciliary localization. Results are expressed as the fraction of Gli2+ cilia with 95% CI. At least 55 ciliated cells were analysed for each condition. *p<0.05, ** p<0.001 (hypothesis test for proportions). (C) The amount of ciliary Gli2 was estimated measuring the GFP fluorescence at the ciliary tip as explained in Materials and Methods. The mean ciliary fluorescence was normalised against the mean total GFP fluorescence of the cell, so as to correct for variations in Gli2 expression among different cells.* p<0.05, *** p<0.0001 (Kruskal Wallis test). (A-C) are representative of four independent experiments. (D-E) NIH/3T3 Flp-In stable cell lines expressing GFP-Gli2 or GFP-Gli2-mutNLS-1+2 were treated with SAG or DMSO as control in the same conditions than those described in (A) and cells were analyzed by confocal microscopy. The amount of nuclear Gli2 (D) was estimated as described in legend of . At least 60 cells were analysed for each condition. *** p<0.0001 (ANOVA). The fraction of Gli2 positive cilia (E) was quantified as described in part (B) of this legend. ** p<0.001 (hypothesis test for proportions).

Article Snippet: The primary antibodies used were anti-Gli2 (H-300 rabbit polyclonal, Santa Cruz Biotechnology, 1:500, or goat polyclonal, R&D Systems (AF3635), 1:1000, overnight at 4°C), anti-GFP (rabbit polyclonal, Life or Cell Signaling Technology, or rat polyclonal, ChromoTek, 1:1000, 2 h at room temperature), anti-Imp-α1(#MAB6207) and Imp-β1 (#MAB8209) (mouse monoclonals, R&D Systems, 1:7000 and 1:1000 respectively, overnight at 4°C), anti-Imp-β2 (mouse monoclonal, Abcam #ab10303, 1:1000, overnight at 4°C) and anti-α tubulin (mouse monoclonal, Sigma, 1:2000, 1 h at room temperature).

Techniques: Expressing, Control, Staining, Fluorescence, Stable Transfection, Confocal Microscopy

Journal: PLoS ONE

Article Title: Ciliary Entry of the Hedgehog Transcriptional Activator Gli2 Is Mediated by the Nuclear Import Machinery but Differs from Nuclear Transport in Being Imp-α/β1-Independent

doi: 10.1371/journal.pone.0162033

Figure Lengend Snippet: (A) NIH/3T3 Flp-In cells expressing GFP-Gli2 were transfected with either myc-MBP-M9M or myc-MBP as control, stimulated with SAG and Gli2 ciliary localization was analysed in transfected cells. Cells were stained for cilium (anti-Ac.Tub, red), GFP-Gli2 (anti-GFP, green), myc-MBP or myc-MBP-M9M (anti-myc, magenta) and nucleus (DAPI, blue). Small pictures show amplification of the selected region. Yellow and white arrows indicate cilia with or without Gli2 at the ciliary tip respectively. Scale bar: 10 μm. (B) Quantification of Gli2 ciliary localization in transfected cells. Results are expressed as the fraction of Gli2 positive cilia in transfected cells with 95% CI. At least 50 cilia from transfected cells were analysed for each sample.* p<0.05, **p<0,001 (hypothesis test for proportions). (C) Fraction of ciliated cells among transfected cells, expressed as 95% CI. At least 120 transfected cells were analysed in each condition. n.s. (not significant) p>0.05 (hypothesis test for proportions). (D) Measurement of cilia length in transfected cells. Each point represents a measurement for a single cilium; red lines represent the median length. At least 60 cilia were measured for each condition. n.s. (not significant) p>0.05 (Mann-Whitney test). (E) WB detecting Imp-β2 after precipitating GFP-Gli2 with GFP-Trap from HEK293FT transfected with pEGFP-Gli2. Membranes were cut at different levels so as to detect in the same samples the precipitated GFP-Gli2 and GFP. The levels of GFP-Gli2, Imp-β2 and GFP in the lysates used for immunoprecipitation were assessed by WB. The band corresponding to GFP looks distorted because the protein migrates with the dye front. (F) Quantification of nuclear Gli2 in transfected cells was performed as described in . At least 60 cells were analysed for each condition. *** p<0.0001 (Kruskal-Wallis test). (A-E) are representative of 3 independent experiments. (G) Activation of a luciferase-based Hh reporter gene in NIH/3T3 transfected with plasmids coding for myc-MBP or myc-MBP-M9M and then stimulated with SAG or DMSO as control. As explained in Materials and Methods, RLU values from SAG treated cells are normalised against the RLU values from non-activated cells and expressed as mean ± s.d from triplicates from two independent experiments. ** p<0.001 (Mann-Whitney test). (H) Quantification of nuclear Gli2 in NIH/3T3 cells transfected with pEGFP-Gli2 alone, or pEGFP-Gli2 plus plasmids coding for myc-MBP or myc-MBP-M9M. Some cells transfected with pEGFP-Gli2 alone were treated with IPZ for 1 hour before activation of the Hh pathway with SAG. In the case of cells tranfected with myc-MBP or myc-MBP-M9M, Gli2 nuclear fluorescence was determined in myc-positive cells. Quantification was performed as described in legend to . Results are representative of two experiments and at least 50 cells were analysed for each condition.* p<0.05, *** p<0.0001 (ANOVA).

Article Snippet: The primary antibodies used were anti-Gli2 (H-300 rabbit polyclonal, Santa Cruz Biotechnology, 1:500, or goat polyclonal, R&D Systems (AF3635), 1:1000, overnight at 4°C), anti-GFP (rabbit polyclonal, Life or Cell Signaling Technology, or rat polyclonal, ChromoTek, 1:1000, 2 h at room temperature), anti-Imp-α1(#MAB6207) and Imp-β1 (#MAB8209) (mouse monoclonals, R&D Systems, 1:7000 and 1:1000 respectively, overnight at 4°C), anti-Imp-β2 (mouse monoclonal, Abcam #ab10303, 1:1000, overnight at 4°C) and anti-α tubulin (mouse monoclonal, Sigma, 1:2000, 1 h at room temperature).

Techniques: Expressing, Transfection, Control, Staining, Amplification, MANN-WHITNEY, Immunoprecipitation, Activation Assay, Luciferase, Fluorescence

Journal: Molecular & cellular proteomics : MCP

Article Title: A New Monoclonal Antibody Enables BAR Analysis of Subcellular Importin β1 Interactomes.

doi: 10.1016/j.mcpro.2022.100418

Figure Lengend Snippet: FIG. 2. Importin β1 BioID. A, structural model of the importins complex with an NLS-bearing cargo protein, nucleoplasmin, from https://pdb1 01.rcsb.org/motm/85. B, schematic showing a set of KPNB1-BioID constructs superimposed on the structural model. Constructs have N- or C-terminal BirA* fusions and Venus reporter with Myc tag on the opposing termini. Pink shading around BirA* indicates 10 nm range of bio- tinylation. C, volcano plots of label-free LC-MS/MS analysis from HeLa cells overexpressing N- or C-terminal BirA* KPNB1 constructs. D, Venn diagram: numbers of significant hits obtained by each construct. E, physical network representation of N- and C-terminal KPNB1-BioID interactomes, based on STRING with a cut-off interaction score of 0.4. KPNB1 is highlighted in red, direct interacting partners in light pur- ple, and second order interactors in gray. IBB, importin β1–binding domain of importin α; GS, Glycine-Serine linker; NLS, nuclear localization signal.

Article Snippet: Expression constructs for KPNB1 were based on pEGFP-N1 human full-length KPNB1 from Addgene (#106941).

Techniques: Construct, Liquid Chromatography with Mass Spectroscopy, Binding Assay

Journal: Molecular & cellular proteomics : MCP

Article Title: A New Monoclonal Antibody Enables BAR Analysis of Subcellular Importin β1 Interactomes.

doi: 10.1016/j.mcpro.2022.100418

Figure Lengend Snippet: FIG. 4. mAbKPNB1-301-320 recognizes importin β1 in adult DRG neurons. A, confocal images of adult DRG neurons in culture, stained for importin β1 using mAbKPNB1-301-320 (mAbKPNB1, 4.6 nM). Middle panel after preincubation with a peptide comprising the epitope sequence (9.5 nM KPNB1301–320), right panel is omitted primary antibody control. The scale bar represents 20 μm. B, quantification of mAbKPNB1-301-320 average fluorescence intensity using ImageJ, Mean ± SEM, n ≥5, one-way ANOVA, ** indicates p < 0.01. C, electron micrographs of ultrathin monolayer sections showing immunogold labeling of importin β1 in cultured mouse DRG neurons using mAbKPNB1-301-320 (mAbKPNB1, 0.1 μg/μl), left and right panels. Middle panel after preincubation with a peptide comprising the epitope sequence (1 μg/μl KPNB1301–320). The scale bars represent 100 nm; gold particle diameter: 10 nm. D, proximity ligation assay (PLA, green) showing complexes of importin β1 (KPNB1) with dynein (Dync1h1) in axons of cultured DRG neurons. Neurofilament heavy chain (NFH, red) and DAPI (blue). Scale bars: full image 50 μm, insert 10 μm. E, quantification of PLA signal in axons, one-way ANOVA with Dunnett’s multiple comparisons test, **** indicates p <0.0001, n > 13. DRG, dorsal root ganglia.

Article Snippet: Expression constructs for KPNB1 were based on pEGFP-N1 human full-length KPNB1 from Addgene (#106941).

Techniques: Staining, Sequencing, Control, Labeling, Cell Culture, Proximity Ligation Assay

Journal: Molecular & cellular proteomics : MCP

Article Title: A New Monoclonal Antibody Enables BAR Analysis of Subcellular Importin β1 Interactomes.

doi: 10.1016/j.mcpro.2022.100418

Figure Lengend Snippet: FIG. 5. mAbKPNB1-301-320 detects importin β1 upregulation in injured nerve. A, immunostaining of cryosections with mAbKPNB1-301- 320 (mAbKPNB1, 2.7 ng/μl) before and 24 h after sciatic nerve crush. The scale bar represents 10 μm. B, quantification of analysis shown in (A) in ImageJ, using NFH staining as a mask for axons. Mean ± SEM, n ≥490 axons, Unpaired two-tailed t test, **** indicates p < 0.0001. C, confocal imaging of PLA. Upper, PLA for importin β1 with mAbKPNB1 and a commercial anti-KPNB1 antibody (MBS); lower: PLA for dynein and importin β1 with anti-Dync1h1 and mAbKPNB1; both on cross sections of the sciatic nerve, the scale bar represents 5 μm. D and E, Quantification of the analyses shown in (C), for importin β1 specific detection (D) or importin β1 in complex with dynein (E). Mean ± SEM, n > 2000 axons, Unpaired two-tailed t test, ** indicates p < 0.01, **** p < 0.0001.

Article Snippet: Expression constructs for KPNB1 were based on pEGFP-N1 human full-length KPNB1 from Addgene (#106941).

Techniques: Immunostaining, Staining, Two Tailed Test, Imaging

Journal: Molecular & cellular proteomics : MCP

Article Title: A New Monoclonal Antibody Enables BAR Analysis of Subcellular Importin β1 Interactomes.

doi: 10.1016/j.mcpro.2022.100418

Figure Lengend Snippet: FIG. 6. BAR characterization of the cytoplasmic importin β1 interactome in adult DRG neurons. A, visualization of mAbKPNB1-301-320– directed BAR reaction in a cultured DRG neuron, showing endogenous cytoplasmic importin β1 (green) and biotinylation (red). Scale bars: full image 40 μm, cell body 10 μm, axon 20 μm. Note the predominantly cytoplasmic and axonal biotinylation. B, volcano plot of proteins identified by mass spectrometry with BAR, multiple t test with a desired false discovery rate of 10%, n = 3. C, network analysis of hits from (B) showing subnetwork of direct binding partners based on STRING database. KPNB1 in red, retrograde motor interactors in green, translation machinery in light blue, nucleocytoplasmic transport in light purple, and other second order interactors in gray. D, partial network of selected enriched terms with focus on the axonal compartment: colored by cluster ID, nodes that share the same cluster ID are closer to each other, generated by Metascape. BAR, biotinylation by antibody recognition; DRG, dorsal root ganglia.

Article Snippet: Expression constructs for KPNB1 were based on pEGFP-N1 human full-length KPNB1 from Addgene (#106941).

Techniques: Cell Culture, Mass Spectrometry, Binding Assay, Generated

Journal: Nucleic Acids Research

Article Title: The pioneer round of translation ensures proper targeting of ER and mitochondrial proteins

doi: 10.1093/nar/gkab1098

Figure Lengend Snippet: The CBC–RNC–SRP complex is translationally repressed. ( A ) Protein expression from Con-FLAG-GPx1 or PPL-FLAG-GPx1 mRNA. HEK293T cells depleted of the indicated protein were transiently transfected with one of reporter plasmids and pMS2-HA-GFP (a reference plasmid). The cells were either untreated or treated with MG132 for 12 h before cell harvesting. The levels of the expressed proteins were normalized to MS2-HA-GFP protein abundance. Efficient inhibition of proteasomal activity by MG132 treatment was evidenced by an increase in the abundance of endogenous p53 through its stabilization; n = 3. ( B ) Protein expression from Myc-ER-GFP mRNAs. As performed in panel A, except that HEK293T cells were transiently transfected with pCMV-Myc-ER-GFP and a reference plasmid expressing λN-HA-GST; n = 3. ( C ) Complementation experiments using CBP80 R -WT or -L34E variant and either Con-FLAG-GPx1 mRNA or PPL-FLAG-GPx1 mRNA. As performed in panel A, except that HEK293T cells, either undepleted or depleted of both SRα and CBP80 were transiently transfected with (i) a plasmid expressing either CBP80 R -WT-HA or CBP80 R -L34E-HA, (ii) a reporter plasmid expressing Con-FLAG-GPx1 or PPL-FLAG-GPx1 mRNA, and (iii) a reference plasmid, λN-HA-GST; n = 3. ( D ) Complementation experiments using CBP80 R -WT or -L34E and Myc-ER-GFP mRNAs. As performed in panel C, except that protein expression from Myc-ER-GFP mRNA was analyzed; n = 3.

Article Snippet: The following plasmids were used in this study: pCMV-Myc and pEGFP-C2 (Clontech); p3×FLAG-CMV™-7.1 (MilliporeSigma); pCMV-Myc-ER-GFP (Invitrogen); pCMV-Myc-GFP ( ); pcDNA3-FLAG, pcDNA3-FLAG-CTIF, pcDNA3-FLAG-CBP80 and pmCMV-GPx1-Norm ( ); pX ( ); pCMV-Myc-eIF4E and pRβGl-SL0-Norm ( ); pcDNA3.1-HA and pλN-HA-GFP ( ); pCXbsr-mRFP-Ub and pCMV-Myc-GST ( ); pMS2-HA-GFP ( ); pCMV3-IMPβ-FLAG (Sino Biological; #HG17676-CF); pOTB7-SRP54, pCMV-SPORT6-SRα, pCMV-SPORT6-DPM3, pOTB7-PAM16, pCMV-SPORT6-SLC25A41 and pOTB7-ATP5F1E (ATP5E; Korea Human Gene Bank; hMU006286, hMU001253, hMU003974, hMU008804, hMU004511 and hMU005976, respectively); Lamp1-RFP (Addgene; #1817), mCh-Sec61β (Addgene; #49155); pcDNA3.1-FLAG-LC3B (a kind gift from Hyun Kyu Song, Korea University, Republic of Korea) and pWT-PPL-TET ( ).

Techniques: Expressing, Transfection, Plasmid Preparation, Cell Harvesting, Inhibition, Activity Assay, Variant Assay

Journal: Science Advances

Article Title: Antiviral activity of a purine synthesis enzyme reveals a key role of deamidation in regulating protein nuclear import

doi: 10.1126/sciadv.aaw7373

Figure Lengend Snippet: ( A ) 293T cells were transfected with plasmids containing FLAG-tagged RTA-WT and the indicated V5-tagged importins. WCLs were incubated with anti-V5 antibody. The precipitated proteins and WCLs were analyzed by immunoblotting with indicated antibodies. ( B ) Glutathione agarose loaded with GST or recombinant GST–importin β1 was incubated with purified RTA. Precipitated proteins and RTA (input) were analyzed by immunoblotting with anti-RTA antibody, while GST and GST–importin β1 were analyzed by Coomassie staining (bottom). ( C ) SLK/iBAC.RTA-WT cells were induced with doxycycline (1 μg/ml) for 24 hours and then transfected with a plasmid containing EGFP-bimax2 for 24 hours. Cells were analyzed by immunofluorescence staining and microscopy. ( D ) Glutathione agarose loaded with GST fusions containing either importin β1 (imp-β1) or β2 (imp-β2) was incubated with WCLs containing RTA-WT (WT) or RTA-DD (DD). Precipitated proteins and WCLs (Input) were analyzed by immunoblotting with anti-RTA antibody (right). GST–importin β1 and GST–importin β2 were analyzed by Coomassie staining. ( E ) 293T cells were transfected with plasmids containing RTA-WT (WT) or RTA-DD (DD) mutant. WCLs were prepared and precipitated with control immunoglobulin G (IgG) or antibody against importin β1 (Imp-β1). Precipitated proteins and WCLs were analyzed by immunoblotting with indicated antibodies. ( F ) iSLK/rKSHV.219 cells were induced with doxycycline (0.5 μg/ml) and sodium butyrate (1 mM) for the indicated times. Immunoprecipitation and immunoblotting were performed as described in (E). ( G ) iSLK/rKSHV.219 cells were transduced with control lentivirus (CTL) or lentivirus encoding shRNA against PFAS, followed by doxycycline and sodium butyrate induction for 72 hours. Cells were harvested for cellular fractionation to obtain cytosolic (C) and nuclear (N) fractions that, along with WCLs, were analyzed by immunoblotting with indicated antibodies. The results shown in (A), (B), and (D) to (G) represent three independent experiments ( n = 3).

Article Snippet: Antibodies against FLAG (M2, Sigma), importin α1 (sc-101292, Santa Cruz Biotechnology), importin β1 (NB100-94993, Novus Biologicals), importin β2 (sc-32314, Santa Cruz Biotechnology), human influenza hemagglutinin (HA) (MMS-101P, BioLegend), goat anti-rabbit/mouse immunoglobulin G (H+L) Alexa Fluor 488 (A-11034 and A32723, Invitrogen), tubulin (DM1A, Cell Signaling), and histone H3 (1B1B2, Cell Signaling) were purchased from the indicated suppliers.

Techniques: Transfection, Incubation, Western Blot, Recombinant, Purification, Staining, Plasmid Preparation, Immunofluorescence, Microscopy, Mutagenesis, Control, Immunoprecipitation, Transduction, shRNA, Cell Fractionation

Journal: Science Advances

Article Title: Antiviral activity of a purine synthesis enzyme reveals a key role of deamidation in regulating protein nuclear import

doi: 10.1126/sciadv.aaw7373

Figure Lengend Snippet: ( A ) Alignment of RTA proteins of KSHV, RRV, EBV, HVS, and MHV68 shows the bipartite NLS and the two deamidation sites corresponding to N37 and N225 of KSHV RTA. ( B ) 293T stable cells carrying control shRNA or PFAS shRNA were transfected with a plasmid containing RRV RTA (rRTA), EBV RTA (eRTA), HVS RTA (hRTA), or MHV68 RTA (mRTA). WCLs were prepared at 30 hours after transfection and analyzed by two-dimensional gel electrophoresis and immunoblotted for RTA (left). WCLs were analyzed by immunoblotting with antibodies against PFAS and RTA (right). ( C ) 293T cells transfected with plasmids containing rRTA, eRTA, hRTA, or mRTA. WCLs were precipitated with a control IgG or antibody against importin β1. Precipitated proteins and WCLs were analyzed by immunoblotting with indicated antibodies. ( D ) Glutathione agarose loaded with GST or GST–importin β1 (GST–imp β1) were incubated with WCLs prepared from 293T cells transfected with a plasmid containing eRTA, hRTA, or mRTA, without or with a plasmid containing PFAS-ED. Precipitated proteins and WCLs were analyzed by immunoblotting with indicated antibodies. ( E ) 293T cells were transfected with wild type (WT) or the deamidated mutant (DD/D) of rRTA, hRTA, or eRTA. Sites of N>D mutations were highlighted in (A). Nuclear (N) and cytosolic (C) fractions were obtained by sequential centrifugation and analyzed by immunoblotting with indicated antibodies. WCLs were analyzed for the expression of RTA wild type and the DD/D mutant (right panels). The results shown in (B) to (E) represent three independent experiments ( n = 3).

Article Snippet: Antibodies against FLAG (M2, Sigma), importin α1 (sc-101292, Santa Cruz Biotechnology), importin β1 (NB100-94993, Novus Biologicals), importin β2 (sc-32314, Santa Cruz Biotechnology), human influenza hemagglutinin (HA) (MMS-101P, BioLegend), goat anti-rabbit/mouse immunoglobulin G (H+L) Alexa Fluor 488 (A-11034 and A32723, Invitrogen), tubulin (DM1A, Cell Signaling), and histone H3 (1B1B2, Cell Signaling) were purchased from the indicated suppliers.

Techniques: Control, shRNA, Transfection, Plasmid Preparation, Two-Dimensional Gel Electrophoresis, Electrophoresis, Western Blot, Incubation, Mutagenesis, Centrifugation, Expressing

Journal: bioRxiv

Article Title: Karyopherin mimicry explains how the HIV capsid penetrates nuclear pores

doi: 10.1101/2023.03.23.534032

Figure Lengend Snippet: Confocal fluorescence microscopy (all channels) with Nup98 condensates and mCherry labelled controls or CA-constructs. a-f , Single z-plane images (cyan, 488-Nup channel; magenta, protein-mCherry channel) of (a) control fusion protein IBBmCherry (b) control complex Importin β:IBBmCherry (c) unassembled CA-mCherry (d) unassembled CA-mCherry carrying FG pocket mutation N57A (e) cross-linked CA hexamer-mCherry (f) cross-linked CA hexamer-mCherry carrying N57A. Background subtracted radially averaged fluorescence intensity across condensates for 488-Nup channel (cyan) and protein-mCherry channel (magenta). Mean intensity curves shown in bold. Scale bar 5 µm (main), 1 µm (inset).

Article Snippet: Importinβ and IBB-mCherry in pET11a (Genscript) were expressed as His-fusion proteins in E. coli Rosetta2 cells.

Techniques: Fluorescence, Microscopy, Construct, Control, Mutagenesis

Journal: bioRxiv

Article Title: Karyopherin mimicry explains how the HIV capsid penetrates nuclear pores

doi: 10.1101/2023.03.23.534032

Figure Lengend Snippet: Confocal fluorescence microscopy (all channels) of Nup98 condensates with Importinβ/mCherry controls. a-b , Single z-plane images (cyan, 488-Nup channel; magenta, protein-mCherry channel) of (a) control mCherry (b) control Importin β and mCherry. Background subtracted radially averaged fluorescence intensity across condensates for 488-Nup channel (cyan) and protein-mCherry channel (magenta). Mean intensity curves shown in bold. Scale bar 5 µm (main), 1 µm (inset).

Article Snippet: Importinβ and IBB-mCherry in pET11a (Genscript) were expressed as His-fusion proteins in E. coli Rosetta2 cells.

Techniques: Fluorescence, Microscopy, Control

Journal: bioRxiv

Article Title: Karyopherin mimicry explains how the HIV capsid penetrates nuclear pores

doi: 10.1101/2023.03.23.534032

Figure Lengend Snippet: Confocal fluorescence microscopy (all channels) of Nup98 condensates and CA-mCherry in the presence of karyopherins Importinα:Importinβ, TNPO-1 and TNPO-3. a-d , Single z-plane images (cyan, 488-Nup channel; magenta, protein-mCherry channel) of (a) CA-mCherry (b) CA-mCherry and Importinα:Importinβ (c) CA-mCherry and TNPO-1 (d) CA-mCherry and TNPO-3. Background subtracted radially averaged fluorescence intensity across condensates for 488-Nup channel (cyan) and protein-mCherry channel (magenta). Mean intensity curves shown in bold. Nup98 signal was reduced in the presence of karyopherins, suggesting that binding of karyopherins to FG-motifs reduced the compactness of the condensates. Selective properties of the condensates were still maintained (compare ). Scale bar 5 µm (main), 1 µm (inset).

Article Snippet: Importinβ and IBB-mCherry in pET11a (Genscript) were expressed as His-fusion proteins in E. coli Rosetta2 cells.

Techniques: Fluorescence, Microscopy, Binding Assay

Journal: bioRxiv

Article Title: Karyopherin mimicry explains how the HIV capsid penetrates nuclear pores

doi: 10.1101/2023.03.23.534032

Figure Lengend Snippet: Confocal fluorescence microscopy (all channels) with Nup98 condensates and CA hexamer -mCherry in the presence of karyopherins Importinα:Importinβ, TNPO-1 and TNPO-3. a-d , Single z-plane images (cyan, 488-Nup channel; magenta, protein-mCherry channel) of (a) CA hexamer-mCherry (b) CA hexamer-mCherry and Importinα:Importinβ (c) CA hexamer-mCherry and TNPO-1 (d) CA hexamer-mCherry and TNPO-3. Background subtracted radially averaged fluorescence intensity across condensates for 488-Nup channel (cyan) and protein-mCherry channel (magenta). Mean intensity curves shown in bold. Nup98 signal was reduced in the presence of karyopherins, suggesting that binding of karyopherins to FG-motifs reduced the compactness of the condensates. Selective properties of the condensates were still maintained (compare ). Scale bar 5 µm (main), 1 µm (inset).

Article Snippet: Importinβ and IBB-mCherry in pET11a (Genscript) were expressed as His-fusion proteins in E. coli Rosetta2 cells.

Techniques: Fluorescence, Microscopy, Binding Assay

Journal: The Journal of Biological Chemistry

Article Title: Augmin is a Ran-regulated spindle assembly factor

doi: 10.1016/j.jbc.2023.104736

Figure Lengend Snippet: Augmin binds to importins. A , structure of the augmin complex, broken into the γ-TuRC binding T-III ( pink ), comprised of subunits Haus1 and Haus3–5, and the MT-binding T-II ( blue ), comprised of subunits Haus2 and Haus6–8. The disordered N terminus of Haus8 (shown as a dashed blue line ) contains the primary MT-binding site. Structure of Xenopus augmin was taken from Ref. . B , glutathione beads bound to either GST (control), GST-importin-α ΔIBB , or GST-importin-β were incubated with full-length augmin, then both the input and bound fraction were Western blotted for intact augmin complexes using an antibody against the Strep-tagged subunits Haus3 (T-III) and Haus8 (T-II). Below, GST and GST-importin loading was demonstrated by Coomassie stain. C , as in ( B ), importin-bound beads were incubated with augmin, either T-III or T-II, and binding of intact augmin subcomplex was detected via Western blot against the Strep-tagged subunits Haus3 and Haus8. D , Haus8 1–150 (fused to an N-terminal Strep-tagged GFP) was incubated with importin-bound beads, and binding was detected via Western blot. E , augmin complex lacking the N-terminal 150 residues of Haus8 was incubated with importin-bound beads, and binding of intact augmin complex was detected via Western blot against augmin subunit Haus1. γ-TuRC, γ-tubulin ring complex; GST, glutathione- S -transferase; IBB, importin-β binding; MT, microtubule; T-III, tetramer III.

Article Snippet: In addition to probing with α-Haus8, pulldown blots were probed using custom antibodies against X. laevis GST-importin-α and GST-importin-β generated by GenScript and purified from serum according to previously published protocols ( , ).

Techniques: Binding Assay, Control, Incubation, Western Blot, Staining

Journal: The Journal of Biological Chemistry

Article Title: Augmin is a Ran-regulated spindle assembly factor

doi: 10.1016/j.jbc.2023.104736

Figure Lengend Snippet: Haus8 of augmin binds to importins and MTs through two NLS sites. A , the augmin subunit Haus8 is predicted to contain two NLS sequences within its disordered N terminus . The domain architecture of Haus8 is cartooned at top , and the sequence of each predicted NLS in Xenopus laevis is shown at the bottom . X. laevis Haus8 is shown aligned to other vertebrate orthologs below, and all basic residues (arginine abbreviated as R and lysine as K) are highlighted in blue . Indicated in red at the top of the sequence are the pairs of basic residues mutated to alanine to generate the Haus8 mutants ΔNLS1 (K27A/K28A) and ΔNLS2 (K143A/K144A). B – E , pulldowns of Strep-GFP-Haus8 1–150 , either wildtype or containing the indicated NLS mutants, were conducted as for Figure 1 D . F , selected TIRF images of in vitro binding of GST-GFP-Haus8 1–150 to stabilized MT seeds. Haus8 constructs with mutated residues in NLS1 and/or NLS2 result in a reduction in binding, as quantified in G . Images belonging to the same experiment were contrast matched. To compare augmin fluorescence intensity across experiments, the intensity was normalized with respect to the tubulin signal. G , boxplot of average GFP-Haus8 signal relative to the average tubulin signal, where each marker represents a single MT from the experiment shown in F . The total number of MTs (n) was collected from two replicates. Center lines show the medians; box limits indicate the 25th and 75th percentiles as determined by R software; whiskers extend 1.5 times the interquartile range from the 25th and 75th percentiles. p Values were calculated from independent t tests. GST, glutathione- S -transferase; MT, microtubule; NLS, nuclear localization signal; TIRF, total internal reflection fluorescence.

Article Snippet: In addition to probing with α-Haus8, pulldown blots were probed using custom antibodies against X. laevis GST-importin-α and GST-importin-β generated by GenScript and purified from serum according to previously published protocols ( , ).

Techniques: Sequencing, In Vitro, Binding Assay, Construct, Fluorescence, Marker, Software

Journal: The Journal of Biological Chemistry

Article Title: Augmin is a Ran-regulated spindle assembly factor

doi: 10.1016/j.jbc.2023.104736

Figure Lengend Snippet: Importins regulate augmin binding to MTs. A , WT GST-GFP-Haus8 localizes strongly to GMPCPP-stabilized MT seeds in vitro ( top row ), as visualized by TIRF microscopy. In the presence of importin-α ΔIBB ( middle row ) or importin-β ( bottom row ), binding of Haus8 to MTs is diminished. This is quantified in B . B , boxplot of average GFP-Haus8 signal relative to the average tubulin signal, where each marker represents a single MT from the experiment shown in F . The total number of MTs (n) was collected from two replicates. The boxes extend from the 25th to 75th percentile, and the upper and lower bars represent the minimum and maximum, respectively. p Values were calculated from independent t tests. C , WT GFP-labeled augmin localized to GMPCPP-stabilized MT seeds in vitro ( top row ), as visualized by TIRF microscopy. In the presence of importin-α ΔIBB ( middle row ) or importin-β ( bottom row ), binding of augmin to MTs is decreased but not eliminated. This is quantified in D . D , boxplot of average GFP-augmin signal relative to the average tubulin signal, where each marker represents a single MT from the experiment shown in F . The total number of MTs (n) was collected from two replicates using two independent augmin preparations. Center lines show the medians; box limits indicate the 25th and 75th percentiles as determined by R software; whiskers extend 1.5 times the interquartile range from the 25th and 75th percentiles. p Values were calculated from independent t tests. In A and C , images belonging to the same experiment were contrast matched. To compare Haus8 fluorescence intensity across experiments, the intensity was normalized with respect to the tubulin signal. Scale bars correspond to 5 μm. GST, glutathione- S -transferase; IBB, importin-β binding; MT, microtubule; TIRF, total internal reflection fluorescence.

Article Snippet: In addition to probing with α-Haus8, pulldown blots were probed using custom antibodies against X. laevis GST-importin-α and GST-importin-β generated by GenScript and purified from serum according to previously published protocols ( , ).

Techniques: Binding Assay, In Vitro, Microscopy, Marker, Labeling, Software, Fluorescence

Journal: The Journal of Biological Chemistry

Article Title: Augmin is a Ran-regulated spindle assembly factor

doi: 10.1016/j.jbc.2023.104736

Figure Lengend Snippet: RanGTP releases importin inhibition of MT binding. A , GFP or GFP-Haus8 1–150 was bound to α-GFP magnetic resin and incubated with importin-αβ in the presence or the absence of a 10-fold excess of Ran Q69L . Both importin-αβ binding and augmin loading were assessed by Coomassie staining and the intensity of bands at the indicated sizes. B , in vitro localization of GST-GFP-Haus8 1–150 binding to GMPCPP-stabilized MT seeds, as visualized by TIRF microscopy ( top row ). Addition of importin-αβ heterodimer inhibits binding of Haus8 to MTs ( middle row ), whereas addition of Ran Q69L rescues MT binding of Haus8 ( bottom row ). Images belonging to the same experiment were contrast matched. To compare Haus8 fluorescence intensity across experiments, the intensity was normalized with respect to the tubulin signal. This is quantified in C . C , boxplot of average GFP-Haus8 signal relative to the average tubulin signal, where each marker represents a single MT from the experiment shown in F . n corresponds to the total number of MTs. Center lines show the medians; box limits indicate the 25th and 75th percentiles as determined by R software; whiskers extend 1.5 times the interquartile range from the 25th and 75th percentiles. p Values were calculated from independent t tests. Scale bars correspond to 5 μm. GST, glutathione- S -transferase; MT, microtubule; TIRF, total internal reflection fluorescence.

Article Snippet: In addition to probing with α-Haus8, pulldown blots were probed using custom antibodies against X. laevis GST-importin-α and GST-importin-β generated by GenScript and purified from serum according to previously published protocols ( , ).

Techniques: Inhibition, Binding Assay, Incubation, Staining, In Vitro, Microscopy, Fluorescence, Marker, Software