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: The Journal of Biological Chemistry

Article Title: Requirement for Lamin B Receptor and Its Regulation by Importin ? and Phosphorylation in Nuclear Envelope Assembly during Mitotic Exit *

doi: 10.1074/jbc.M110.102368

Figure Lengend Snippet: LBR1–210 interacts with importin β at mitosis and dissociates at the end of mitosis. A, HeLa cells, transiently transfected with GFP-xLBR1–210, were co-stained with anti-importin β and DAPI for DNA. Note that this truncate LBR localizes to the nucleus in interphase and moves to mitotic spindle and other cytoplasmic place where it co-localizes with importin β in mitosis. B, co-immunoprecipitation of importin β with GFP-xLBR. The clarified xLBR1–210-expressing cells in both interphase and mitosis were lysed and incubated with a rabbit anti-GFP or rabbit-IgG (as control) antibody for 60 min on ice. Then 15 μl of protein A- or G-Sepharose (75% slurry) were added, and the mixtures were rotated for 2 h at 4 °C. The beads were washed three times with lysis buffer and harvested by brief centrifugation and finally suspended in gel sample buffer. The protein samples were separated by SDS-PAGE gel and processed for Western blot with anti-importin β or anti-GFP antibody. Note that the binding of the truncate GFP-xLBR1–210 with importin β occurred only in mitosis. C, HeLa cells were arrested by double thymidine block/release to various cell cycle phases and lysed to generate time-course samples, and the time at the metaphase onset is referred to as 0 min. The cell lysates were processed for GST pulldown assay between GFP-xLBR1–210 and GST-importin β45–462 using an anti-GFP antibody, followed by Western blot. D, HeLa cells were arrested by thymidine/nocodazole block at metaphase and released. Cell lysates were prepared at the time indicated after release from metaphase and GST pulldown assay between GFP-xLBR1–210 and GST-importin β45–462 was performed using anti-GFP antibody. The lysate GFP-LBR1–210 in GST pulldown assay was used as control. Note that the interaction of GFP-LBR1–210 became weaker along with the release from metaphase.

Article Snippet: Gel Electrophoresis and IB After being separated on 10% SDS-PAGE gels, the protein samples were transferred onto nitrocellulose filters in the transfer buffer (25 m m Tris, 192 m m glycine, and 20% methanol) for 1 h at 100 V. The filters were blocked in TTBS (20 m m Tris-HCl (pH 7.4), 500 m m NaCl, and 0.3% Tween 20) containing 5% nonfat milk for 1 h at room temperature and probed with anti-GFP monoclonal antibody (Santa Cruz Biotechnology, diluted 1:1000 in TTBS with 5% nonfat milk) or probed with anti-importin β monoclonal antibody (BD Transduction, diluted 1:1000 in TTBS with 5% nonfat milk) overnight at 4 °C.

Techniques: Transfection, Staining, Immunoprecipitation, Expressing, Incubation, Lysis, Centrifugation, SDS Page, Western Blot, Binding Assay, Blocking Assay, GST Pulldown Assay

Journal: The Journal of Biological Chemistry

Article Title: Requirement for Lamin B Receptor and Its Regulation by Importin ? and Phosphorylation in Nuclear Envelope Assembly during Mitotic Exit *

doi: 10.1074/jbc.M110.102368

Figure Lengend Snippet: Phosphorylation of LBR Ser-71 is required for the interaction of LBR with importin β, the NE precursor membrane vesicle targeting to the chromatin, and the NE assembly. A, schematic representation of the importin β truncates and their respective binding ability to GFP-xLBR1–210. The gray zone indicates the minimal length (aa 45–462) of the truncated importin β proteins necessary for binding with LBR. B, GST pulldown assay between the GST-importin β proteins and GFP-xLBR1–210. 5 μg of soluble GST or GST-importin β proteins bound to 15 μl of glutathione-Sepharose beads (75% slurry) was incubated with lysate of HeLa cells expressing GFP-xLBR1–210 for 3 h at 4 °C. The bound proteins of the beads were processed for Western blot using an anti-GFP antibody. Note that importin β45–462 was the minimum size to bind with GFP-xLBR1–210, and any truncate containing this fragment such as importin β1–462 and importin β45–876 as well as the full-length importin β1–876 could bind with GFP-xLBR1–210 equally. C, part of xLBR amino acid sequence, showing the serine-rich conserved region (SR-rich region). D, pulldown assay using GST-importin β45–462-coated beads and mitotic HeLa cell lysates expressing different GFP-xLBR truncated proteins or GFP as control, followed by a Western blot analysis with an anti-GFP antibody. Note that the aa 69–90 region of xLBR is required for its binding with importin β45–462 and its serine 71 is critical for this binding. E, pulldown assay using GST-importin β45–462-coated beads and the lysate of mitotic HeLa cells expressing the wild-type truncate GFP-xLBR1–210, the unphosphorylation-mimicking mutant GFP-xLBR1–210-S71A or the phosphorylation-mimicking mutant GFP-xLBR1–210-S71D, or GFP alone as control, followed by a Western blot analysis with an anti-GFP antibody. Note that importin β45–462 only bound with wild-type GFP-xLBR1–210 and the serine 71 phosphorylation-mimicking mutant GFP-xLBR1–210-S71D. F, HeLa cells expressing the wild-type GFP-LBR, GFP-LBR1–621-S71D, and GFP-xLBR1–210-S71A were fixed and count-stained with DAPI. Images were collected using a Zeiss immunofluorescence microscope 200M equipped with a 63× objective and a cooled charged-coupled device AxioCamMRm camera. Bars, 10 μm. G, phosphorylation site identification of the peptide (64KGGSTSSSPSR) by nanoLC-MS/MS from in-gel tryptic digest of hLBR. MS/MS fragmentation of the doubly charged ion m/z 565.7467 gave a sequential b and y ion sequence to match the sequence of one peptide from hLBR (64KGGSTSSSPSR, [M+H]+ 1130.4861 Da) with a phosphate modification. The b and y ions showed that the phosphorylation site was located at the serine 71 position.

Article Snippet: Gel Electrophoresis and IB After being separated on 10% SDS-PAGE gels, the protein samples were transferred onto nitrocellulose filters in the transfer buffer (25 m m Tris, 192 m m glycine, and 20% methanol) for 1 h at 100 V. The filters were blocked in TTBS (20 m m Tris-HCl (pH 7.4), 500 m m NaCl, and 0.3% Tween 20) containing 5% nonfat milk for 1 h at room temperature and probed with anti-GFP monoclonal antibody (Santa Cruz Biotechnology, diluted 1:1000 in TTBS with 5% nonfat milk) or probed with anti-importin β monoclonal antibody (BD Transduction, diluted 1:1000 in TTBS with 5% nonfat milk) overnight at 4 °C.

Techniques: Binding Assay, GST Pulldown Assay, Incubation, Expressing, Western Blot, Sequencing, Mutagenesis, Staining, Immunofluorescence, Microscopy, Tandem Mass Spectroscopy, Modification