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: 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: medRxiv

Article Title: Genetic SNUPN variants cause spinocerebellar atrophy by disrupting global splicing in Purkinje cells

doi: 10.1101/2024.07.11.24310169

Figure Lengend Snippet: (a) T1-weighted sagittal midline images of a control individual (Control), Patient 1, Patient 2, and Patient 3.at age range of 10-15 years-old. Images of all patients show marked dilation of the cerebellar interfolia spaces and a marked decrease in size of the superior and inferior vermis (arrows). (b) The inheritance pattern of affected individuals is consistent with a fully penetrant autosomal recessive disorder. Squares and circles indicate males and females, respectively. Black-filled and open symbols indicate affected individuals a nd unaffected relatives, respectively. * denotes individual enforced whole exome sequencing (WES). (c) The location of identified snurportin-1 variants. Regions related to import/export into/from the nucleus are shown in orange and blue, r espectively. R55W is located at the binding domain with importinβ, and R204Q is located in the CRM1 binding domain. (d) Localization of wt and mutant GFP-snurportin-1 in HeLa cells after treatment with nuclear import and export inhibitors. Scale bar, 50 μm.

Article Snippet: Binding of the snurportin-1 fusion proteins and recombinant human importin beta (Novus Biologicals) was measured by following a series of different importin beta concentrations (7.4, 22.2, 66.7, 200, and 600 nM) to the snurportin-1 fragments (2 μg/ml) fixed on a sensor chip with glutathione S-transferase antibodies.

Techniques: Control, Sequencing, Binding Assay, Mutagenesis

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: bioRxiv

Article Title: SV40 exploits the Nesprin-2-SUN1-KPNA4 axis for stepwise targeting and entry into the host nucleus to promote infection

doi: 10.64898/2026.03.15.711898

Figure Lengend Snippet: (A) CV-1 cells transfected with the indicated siRNA were infected with SV40 and the resulting whole cell extracts were subjected to SDS-PAGE followed by immunoblotting. The extent of depletion by the KPNA1, KPNA2, and KPNA4 siRNA is shown in the Western blots below. Data were normalized to the Scr control. (B) The T-Ag band intensity in A was quantified by the FIJI software. Data were normalized to the Scr control. (C) CV-1 cells transfected with the indicated siRNA were infected with SV40 and the resulting whole cell extracts were subjected to SDS-PAGE and immunoblotting. (D) The T-Ag band intensity in C was quantified by the FIJI software. Data were normalized to the Scr control. (E) CV-1 cells were fixed and stained for Nesprin-2 (red) and either one of the KPNA1, KPNA2, or KPNA4 protein (green) and counterstained with DAPI (blue). Scale bar: 10 µm. (F) Pearson’s coefficient was used to quantify colocalization between KPNA1, KPNA2, or KPNA4 with Nesprin-2. Each data point represents one field of view with at least 10 cells. * p ≤ 0.05; ** p ≤ 0.01; *** p ≤ 0.001

Article Snippet: Antibodies, along with the companies that were purchase from and the corresponding catalog numbers, are indicated: SUN1 (Invitrogen, MA5-47231), SUN2 (Proteintech, 27556-1-AP), β actin (Cell Signaling, 4967S), SV40 large T-antigen (Western blot: Santa Cruz Biotechnology, SC-53448; Immunofluorescence: Santa Cruz Biotechnology, SC-147), Nesprin-2 (IP and Immunofluorescence: Invitrogen, MA5-18075; Western blot: Bethyl, A305-393A), SV40 VP1 (Abcam, ab53977), SV40 VP2/3 (Abcam, ab53983), Bap31 (Invitrogen, MA 3002), Hsp90 (Santa Cruz Biotechnology, sc-13119), M2 FLAG (Millipore, F3165-1MG), FLAG (Millipore, F7425), BicD2 (Abcam, ab117818), KPNA1 (Proteintech, 18137-1-AP), KPNA2 (Invitrogen, 108191AP150UL), KPNA4 (Invitrogen, PA518239), Mab414 (Abcam, ab24609).

Techniques: Transfection, Infection, SDS Page, Western Blot, Control, Software, Staining

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