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: Fig. 1. Effect of substitution of lysine residues in the Ku70 NLS with ace tyl–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 pre sented 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: 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: Communications Biology

Article Title: Ibetazol, a novel inhibitor of importin β1-mediated nuclear import

doi: 10.1038/s42003-024-07237-8

Figure Lengend Snippet: A Rationale for the importin α1 localization screen. Importin α1 acts as adapter protein between importin β1 and its cargos and, in steady state, it resides predominantly in the nucleus. Compounds that interfere with the importin β1-importin α1 interaction will result in importin α1 cytoplasmic accumulation. B Left: Localization of importin α1-mNeonGreen in endogenously tagged HeLa Kyoto cells upon knockdown of KPNB1 or upon treatment with 50 µM CDD-01 for 2 h as compared to vehicle control (DMSO). Right: Untagged importin α1 shows similar relocalization to the cytoplasm upon compound treatment as confirmed by immunofluorescence (IF) staining in wild type cells. mNGreen: mNeonGreen tag; AF488: Alexa Fluor™ 488 label. Bar: 20 µm. C Overview of the applied screening cascade. A primary screen (single dose) based on importin α1-mNeonGreen localization in Hela Kyoto cells is followed by dose-response confirmation and chemical validation (independent resynthesis) of the initial hits in the same cell line. One chemical series was further characterized in three importin β1 dependent import assays (translocation of NLS cMyc dependent mNeongreen reporter, p65 and SREBP1-mNeongreen) and two counter screens (exportin-1 dependent export and transportin-1/2 dependent import). D Chemical structure of compounds CDD-01, CDD-02, ibetazol (CDD-03) and CDD-04. E Dose-response curve for importin α1-mNeonGreen relocalization upon 2 h compound treatment. %HIGH: percentage of cells with predominant nuclear localization of mNeonGreen (ratio of intensity in nucleus over cytoplasm is >1.4). Curves where fit by non-linear regression using GraphPad Prism, n = 5 wells from 2 independent experiments. Inset: treatment with reference compound importazole (IPZ) at 50 µM for 2 h does not induce relocalization of importin α1-mNeonGreen to the cytoplasm.

Article Snippet: Adenoviral mediated knockdown of KPNB1 employing Ad-U6-h- KPNB1 -shRNA (Vector Biolabs), which results in clear cytoplasmic relocalization of the importin α1-mNeonGreen fusion protein, was used as a positive control.

Techniques: Knockdown, Control, Immunofluorescence, Staining, Biomarker Discovery, Translocation Assay

Journal: Journal of Cell Science

Article Title: RNA-binding protein HuR regulates nuclear import of protein

doi: 10.1242/jcs.192096

Figure Lengend Snippet: HuR stabilizes mRNA for importin-β1 (Kpnb1) but not for importin-α1. (A,B) Immunoblots of importin-α1 in M2−/− (A) or MCF-7 (B) cells stably expressing shCtrl or shHuR. (C,D) Immunoblots of importin-β1 in M2−/− (C) or MCF-7 (D) cells stably expressing shCtrl or shHuR. (E) Levels of mRNA for importin-β1 (Kpnb1) in M2−/− cells stably expressing shCtrl or shHuR, as measured by Q-PCR and normalized to 18s (mean±s.d.; n=3). *P<0.05, paired Student's t-test. (F) M2−/− cells stably expressing shCtrl or shHuR were treated with actinomycin D (2.5 µg/ml). Levels of Kpnb1 mRNA at various time points following treatment were measured by Q-PCR. Data (mean±s.e.m.; n=3) were normalized to corresponding values at time zero. (G) HuR was immunoprecipitated from M2−/− cells. Kpnb1 mRNA that co-precipitated with HuR was detected by semi-quantitative PCR.

Article Snippet: Real-time quantitative PCR Q-PCR was performed using a StepOnePlus real-time PCR system with the following TaqMan probes: Elavl1 , Mm00516012_m1; Kpna1 , Mm00434700_m1; Kpnb1 , Mm00434318_m1; Tnpo1 , Mm00839059_g1; Tnpo2 , Mm00520392_m1; Ranbp1 , Mm00650862_m1; Cyp26a1 , Mm00514486_m1; and 18s rRNA (4352930E, Applied Biosystems).

Techniques: Western Blot, Stable Transfection, Expressing, Immunoprecipitation, Real-time Polymerase Chain Reaction

Journal: PLoS ONE

Article Title: N-terminally truncated POM121C inhibits HIV-1 replication

doi: 10.1371/journal.pone.0182434

Figure Lengend Snippet: (A) Immunoblot analysis showing GST-POM121C (801–987) bound to KPNB1 in HEK293 cells. Cell lysates were analyzed by immunoblotting either directly (left panels: input) or following GST pull-down experiments. Purified GST or GST-POM121C (801–987) complexes were immunoblotted using anti-GST or anti-KPNB1 antibodies. (B) Schematic presentation of POM121C mutants fused with HA-tagged GST. (C) Immunoblot analysis of HEK293 cells expressing truncated forms of POM121C mutants fused with HA-tagged GST. Whole-cell lysates were immunoblotted using anti-HA or anti- α -tubulin antibodies. (D) Effect of GST-POM121C mutants on viral infectivity. HEK293 cells stably expressing GST or the indicated GST-POM121C mutants were infected with VSV-G/NL4-3luc. Luciferase activity was measured 24 h after infection. Relative luciferase activities are shown as ratios (%) of the RLU of EV control cells with standard deviations calculated from three independent experiments. (E) Cell lysates were analyzed by immunoblotting with anti-HA and anti-KPNB1 antibodies either directly (left panels: input) or following GST pull-down experiments (right panels: GST pull-down). (F) Immunoblot analysis of HEK293 cells expressing truncated forms of GST-POM121C mutants. Experiments were done as described in (C). (G) Effects of the indicated GST-POM121C mutants on viral infectivity. Experiments were done as described in (D). Relative luciferase activities are shown as ratios (%) of the RLU of EV control cells with standard deviations calculated from three independent experiments. (H) GST pull-down analysis showing GST-POM121C (801–987) bound to intracellular KPNB1. Cell lysates were analyzed by immunoblotting either directly (left panels: Input) or subsequent to GST pull-down (right panels) with anti-HA (lower panels) or anti-KPNB1 (upper panels) antibodies. Statistical significance was determined by one-way analysis of variance (ANOVA) with Dunnett’s multiple comparison test (D and G). ns, not significant ( P >0.05); * P <0.05, ** P <0.01, *** P <0.001.

Article Snippet: Samples were subjected to SDS-PAGE, transferred to PVDF membranes, and reacted with rat monoclonal antibody to HA (#11867423001, Sigma-Aldrich Co., St. Louis, MO), mouse monoclonal antibody to FLAG [29–22381, Wako Pure Chemical Industries, Ltd., Osaka, Japan], mouse monoclonal antibody to HIV-1 p24 (#ab9071, Abcam, Inc., Cambridge, MA), goat polyclonal antibody to HIV-1 gp120 (#ab21179, Abcam, Inc., Cambridge, MA), HIV-1-positive pooled serum from infected individuals (subtype B) [ ], mouse monoclonal antibody to α-tubulin (#T6199, Sigma-Aldrich Co, St. Louis, MO), rabbit polyclonal antibody to CYPA (#BML-SA296, Enzo Life Sciences, Inc., Farmingdale, NY), or mouse monoclonal antibody to KPNB1 (#GTX22811, GeneTex, Inc., Irvine, CA).

Techniques: Western Blot, Purification, Expressing, Infection, Stable Transfection, Luciferase, Activity Assay, Control, Comparison

Journal: PLoS ONE

Article Title: N-terminally truncated POM121C inhibits HIV-1 replication

doi: 10.1371/journal.pone.0182434

Figure Lengend Snippet: Cellular proteins bound to GST-POM121C (801–987) identified by mass spectrometry.

Article Snippet: Samples were subjected to SDS-PAGE, transferred to PVDF membranes, and reacted with rat monoclonal antibody to HA (#11867423001, Sigma-Aldrich Co., St. Louis, MO), mouse monoclonal antibody to FLAG [29–22381, Wako Pure Chemical Industries, Ltd., Osaka, Japan], mouse monoclonal antibody to HIV-1 p24 (#ab9071, Abcam, Inc., Cambridge, MA), goat polyclonal antibody to HIV-1 gp120 (#ab21179, Abcam, Inc., Cambridge, MA), HIV-1-positive pooled serum from infected individuals (subtype B) [ ], mouse monoclonal antibody to α-tubulin (#T6199, Sigma-Aldrich Co, St. Louis, MO), rabbit polyclonal antibody to CYPA (#BML-SA296, Enzo Life Sciences, Inc., Farmingdale, NY), or mouse monoclonal antibody to KPNB1 (#GTX22811, GeneTex, Inc., Irvine, CA).

Techniques: