Journal:

Article Title: Integration of HIV-1 caused STAT3-associated B cell lymphoma in an AIDS patient

doi: 10.1016/j.micinf.2007.09.008

Figure Lengend Snippet: (A) Sequence of the HIV-1 5’-LTR (upper panel) and 3’-LTR (lower panel) insertion site in the lymphoma genome. Whole sequences of PCR products are registered as GenBank DQ355432 (5’-LTR, 190 bp) and DQ117603 (3’-LTR, 1.5 kbp), respectively. The sequence of the lymphoma genome is shown in the lower line in black letters. The upper colored line indicates the HIV-1 LTR sequence (blue, GenBank K03455 or AF538307) and STAT3 genomic sequence (violet, GenBank AY572796). HIV-1 intervening sequence between 5’LTR and gag is indicated by green. Duplication of the cellular 5 bp (GAATC) and additional dinucleotides (TG in 5’-LTR and CA in 3’-LTR) by HIV-1 integrase are underlined. DraI site is indicated by italics. (B) PCR for the junction region of 3’LTR and STAT3 gene using HIV3LTR-F and Stat3intron-R primers (see Fig. 3D). 1, PBMCs from a healthy donor; 2, HIV-1-positive Molt4 cell line; 3, lymphoma cells with HIV-1integration; 4, KS lesion from the patient; 5, AIDS-related lymphoma from an unrelated patient; 6, lymphoma from a non-HIV-1infected patient; 7, BCBL-1 (KSHV-positive B cell line); 8, No DNA. The lower panel shows the results of an internal control (β-globin gene). (C) PCR of genomic DNA with a STAT3-intron forward primer (F in this figure, Stat3-intronF2) in combination with 5’ LTR reverse primers (lanes 1–6, 55R, 78R, 348R, 495R, 563R and 612R), and a reverse primer positioning between 5’LTR and gag (lane 7, 676R). The upper panel shows the positions of these primers. A 188 bp product was identified when the 676R primer was used with the STAT3 intron primer (lane 7). If the 5’LTR was intact, the predicted size of this amplicon would have been 777 bp. (D) Map of the defective HIV-1 insertion site in the STAT3 gene. Violet numbers indicate the number in GenBank AY572796 (STAT3). Blue boxes are HIV-1 genomes.

Article Snippet: The cells were transfected with STAT3 expression vector by Nucleofector (Amaxa, Cologne, Germany) using O-06 program.

Techniques: Sequencing, Amplification

Journal:

Article Title: Integration of HIV-1 caused STAT3-associated B cell lymphoma in an AIDS patient

doi: 10.1016/j.micinf.2007.09.008

Figure Lengend Snippet: (A) Probe-primer sets for real time PCR. The top line with boxes is a genome map around HIV-1 integration site of HIV-1 3’LTR. Numbers with plus and minus under the genome map indicate distances (bp) from the integration site. Arrows and heavy lines are probe-primer sets of real time PCR. (B) Copy numbers of HIV-1 integration site and STAT3 gene. Black, gray and white bars indicate mean copy number per 100 ng DNA of this case, HIV-1-positive Molt4 cell line, and TY-1 (HIV-1-negative, KSHV-positive B cell line), respectively. Copy numbers per 100 ng DNA are indicated on the top of each bar. Error bars indicate standard errors of triplicate samples.

Article Snippet: The cells were transfected with STAT3 expression vector by Nucleofector (Amaxa, Cologne, Germany) using O-06 program.

Techniques: Real-time Polymerase Chain Reaction

Journal:

Article Title: Integration of HIV-1 caused STAT3-associated B cell lymphoma in an AIDS patient

doi: 10.1016/j.micinf.2007.09.008

Figure Lengend Snippet: (A) Promoter activity of HIV-1 3’LTR by reporter assay. Schematic representation of promoter constructs used in transient transfection assays is shown on the left. Forty-eight hours after transfection, cells were collected and the luciferase activity was measured. The percentage relative luminescence units (RLU) were calculated by dividing firefly activity by renilla activity. Horizontal bars indicate standard deviations of three independent experiments. (B and C) No methylation in a promoter enhancer region of HIV-1 3’LTR in the HIV-1-integrated lymphoma. (B) CpG sites in the promoter enhancer region of 3’LTR of the HIV-1 provirus in the patient with HIV-1-integrated lymphoma (218-529 in GenBank DQ117603). CpG sites are in boldface and numbered from the 5’ end of the LTR (1–11). Nuclear factor-κB and Sp1 sites identified with Motif Search (Kyoto University Bioinfomatics center, Kyoto, Japan, http://motif.genome.jp/) at a 75% cut-off value are indicated by boxes with broken and solid lines, respectively. Sequences used for primers are indicated by underlining. (C) Levels of CpG methylation of the promoter enhancer region of HIV-1 3’LTR in the HIV-1-integrated lymphoma and lymph nodes in the patient. Results of bisulfite genomic sequencing coupled with TA cloning are shown. The methylation status of 10 clones for each sample is presented; methylation of each CpG site is expressed as a filled circle, and unmethylated sites are shown as open circles. Top, schematic description of CpG sites in the 3’LTR of (B). (D-F) Immunohistochemistry of STAT3. The HIV-1-integrated lymphoma cells expressed STAT3 predominantly in the nucleus (D), however, signals of STAT3 were weak and localized in the cytoplasm in the other case of KSHV-positive, AIDS-related lymphoma (E), and were very weak in a case of EBV-positive, AIDS-related lymphoma (F). Original magnification is x400.

Article Snippet: The cells were transfected with STAT3 expression vector by Nucleofector (Amaxa, Cologne, Germany) using O-06 program.

Techniques: Activity Assay, Reporter Assay, Construct, Transfection, Luciferase, Methylation, CpG Methylation Assay, Genomic Sequencing, TA Cloning, Clone Assay, Immunohistochemistry

Journal:

Article Title: Integration of HIV-1 caused STAT3-associated B cell lymphoma in an AIDS patient

doi: 10.1016/j.micinf.2007.09.008

Figure Lengend Snippet: STAT3 expression in AIDS-related and unrelated lymphoma.

Article Snippet: The cells were transfected with STAT3 expression vector by Nucleofector (Amaxa, Cologne, Germany) using O-06 program.

Techniques: Expressing

Journal:

Article Title: Integration of HIV-1 caused STAT3-associated B cell lymphoma in an AIDS patient

doi: 10.1016/j.micinf.2007.09.008

Figure Lengend Snippet: (A) STAT3 expression in the STAT3-transfected TY-1, a KSHV-positive B cell line. The cells were transfected with STAT3 expression vector by Nucleofector (Amaxa, Cologne, Germany) using O-06 program. STAT3 expression was detected by anti-STAT3 mouse monoclonal antibody (green in left panel) and anti-6xHis antibody, followed by Alexa 488-conjugated anti-mouse IgG antibody (Molecular probe, green in right panel). Red color indicates nuclear counterstaining of propidium iodide. (B) Localization of transfected STAT3 in TY-1. His-tagged STAT3 was detected by anti-6x His antibody in the cytoplasm of B cells (left panel). In the presence of IL-6 (Peprotech, Rocky Hill, NJ, 0.1 ng/ml), transfected STAT3 localizes in the nucleus predominantly (right panel). (C) Cell proliferation assay for STAT3-transfected primary B lymphocytes. Primary B cells were isolated from PBMC. The purity of B cell (CD19+) was >95%. The cells were transfected with STAT3 expression vector expressing STAT3 and CD4 by Nucleofector using U-15 program. Transfection efficiency to primary B cells was around 20%. To increase the proportion of transfected cells, the transfected B cells were separated with CD4 microbeads after 16 hours of the transfection (Miltenyl Biotec, Auburn CA). 48 hours after transfection of STAT3 or vector to primary B cells, the proliferation rate was measured with BrdU ELISA (Roche). Raji is an EBV-positive Burkitt lymphoma cell line (untransfected). Numbers in Y-axis indicates absorbance in ELISA. Error bars indicate standard errors of 8 independent experiments.

Article Snippet: The cells were transfected with STAT3 expression vector by Nucleofector (Amaxa, Cologne, Germany) using O-06 program.

Techniques: Expressing, Transfection, Plasmid Preparation, Proliferation Assay, Isolation, Enzyme-linked Immunosorbent Assay

Journal: Viruses

Article Title: Interaction of the Mouse Polyomavirus Capsid Proteins with Importins Is Required for Efficient Import of Viral DNA into the Cell Nucleus

doi: 10.3390/v10040165

Figure Lengend Snippet: In vivo interactions between importin β1 and viral particles at early times post infection. Western blots of 3T6 cell lysates (labeled as Input) or complexes precipitated through antibody against importin β1 (labeled as IP) were performed with antibody against importin β1 ( A ), VP1 ( B ) or VP2/VP3 ( C , D ). As a negative control (NC), samples precipitated through nonspecific anti-mouse IgG were used. The viral DNA was isolated from complexes and amplified by PCR at indicated times post infection ( E ).

Article Snippet: Mouse monoclonal anti-VP2/3, rabbit polyclonal anti-VP1 antibody, (produced in our laboratory), mouse monoclonal anti-VP1, rat monoclonal anti-large T antigen (LT) (provided by B. E. Griffin, Imperial College of Science, Technology and Medicine at St. Mary’s, London, UK), mouse monoclonal anti-importin β1 antibody (Fisher Scientific), rabbit anti-importin β1 antibody (Bioss Antibodies, Woburn, MA, USA), normal mouse IgG (Upstate Biotechnology, Lake Placid, NY, USA), normal rabbit IgG (Upstate Biotechnology), Alexa Fluor ® 488 donkey anti-mouse IgG (Thermo Fisher Scientific), Alexa Fluor ® 488 goat anti-rat IgG (Thermo Fisher Scientific), Alexa Fluor ® 546 donkey anti-rabbit IgG (Thermo Fisher Scientific), goat anti-rabbit IgG-HRP (Bio-Rad), and goat anti-mouse IgG-HRP (Bio-Rad).

Techniques: In Vivo, Infection, Western Blot, Labeling, Negative Control, Isolation, Amplification

Journal: Viruses

Article Title: Interaction of the Mouse Polyomavirus Capsid Proteins with Importins Is Required for Efficient Import of Viral DNA into the Cell Nucleus

doi: 10.3390/v10040165

Figure Lengend Snippet: Proximity ligation assay of VP1 and importin β1 at 6 and 8 hpi. ( A ) PLA assay was performed on 3T6 cells infected with MPyV (200 virus particles per cell), using primary mouse and rabbit antibodies against VP1 or mouse antibody against VP1 and rabbit antibody against importin β1. Next, the oligoprobes tagged anti-mouse and anti-rabbit antibodies were used. Red spots represent the products of amplification after oligonucleotide ligation. DNA was stained by DAPI. As controls, the infected cells were stained with two antibodies (mouse and rabbit) against VP1 and non-infected cells were stained with anti-VP1 and anti-importin β1 antibodies. At the top of each image, the average numbers of the PLA spots quantified in three independent experiments are presented (for each experiment 50 cells were analyzed). The pictures were taken 20× magnification; ( B ) The graph represents the mean values of three independent experiments ± SD. Samples were compared by the Student’s t -test. p values are given and asterisks represent statistically significant differences (** p ≤ 0.01).

Article Snippet: Mouse monoclonal anti-VP2/3, rabbit polyclonal anti-VP1 antibody, (produced in our laboratory), mouse monoclonal anti-VP1, rat monoclonal anti-large T antigen (LT) (provided by B. E. Griffin, Imperial College of Science, Technology and Medicine at St. Mary’s, London, UK), mouse monoclonal anti-importin β1 antibody (Fisher Scientific), rabbit anti-importin β1 antibody (Bioss Antibodies, Woburn, MA, USA), normal mouse IgG (Upstate Biotechnology, Lake Placid, NY, USA), normal rabbit IgG (Upstate Biotechnology), Alexa Fluor ® 488 donkey anti-mouse IgG (Thermo Fisher Scientific), Alexa Fluor ® 488 goat anti-rat IgG (Thermo Fisher Scientific), Alexa Fluor ® 546 donkey anti-rabbit IgG (Thermo Fisher Scientific), goat anti-rabbit IgG-HRP (Bio-Rad), and goat anti-mouse IgG-HRP (Bio-Rad).

Techniques: Proximity Ligation Assay, Infection, Virus, Amplification, Ligation, Staining

Journal: Viruses

Article Title: Interaction of the Mouse Polyomavirus Capsid Proteins with Importins Is Required for Efficient Import of Viral DNA into the Cell Nucleus

doi: 10.3390/v10040165

Figure Lengend Snippet: Effects of mutations in the NLS of VP1 and VP2/3 on viral infection and on binding of viruses to importin β1. ( A ) 3T6 cells were infected with wt or mutant viruses 1, 2, or 3 (using virus equivalent representing 1.5 × 10 4 genomes per cell). Cells were fixed at 24 hpi and LT was stained by specific antibody. The presented data correspond to the mean of three independent experiments +/− standard deviation (s.d.). At least 300 cells were counted per experiment. Samples were compared by the t -test. p values are given and asterisks represent statistically significant differences (* p ≤ 0.1, ** p ≤0.01 *** p ≤ 0.001); ( B ) PLA assay was performed in 3T6 cells at 6 hpi with wt MPyV or the mutant 2 virus (200 virus particles per cell). For this experiment, we used primary mouse and rabbit antibodies against VP1 or mouse antibody against VP1 and rabbit antibody against importin β1. Next, the oligoprobes tagged anti-mouse and anti-rabbit antibodies were used. Red spots represent the products of amplification after oligonucleotide ligation. DNA was stained by DAPI. As controls, the infected cells were stained with two antibodies (mouse and rabbit), both against VP1 and non-infected cells were stained with anti-VP1 and anti-importin β1 antibodies. At the top of each image, the average numbers of the PLA spots quantified in two independent experiments are presented (for each experiment spots in 50 cells were counted). The pictures were taken 20× magnification; ( C ) The graph represents the mean values of two independent PLA experiments.

Article Snippet: Mouse monoclonal anti-VP2/3, rabbit polyclonal anti-VP1 antibody, (produced in our laboratory), mouse monoclonal anti-VP1, rat monoclonal anti-large T antigen (LT) (provided by B. E. Griffin, Imperial College of Science, Technology and Medicine at St. Mary’s, London, UK), mouse monoclonal anti-importin β1 antibody (Fisher Scientific), rabbit anti-importin β1 antibody (Bioss Antibodies, Woburn, MA, USA), normal mouse IgG (Upstate Biotechnology, Lake Placid, NY, USA), normal rabbit IgG (Upstate Biotechnology), Alexa Fluor ® 488 donkey anti-mouse IgG (Thermo Fisher Scientific), Alexa Fluor ® 488 goat anti-rat IgG (Thermo Fisher Scientific), Alexa Fluor ® 546 donkey anti-rabbit IgG (Thermo Fisher Scientific), goat anti-rabbit IgG-HRP (Bio-Rad), and goat anti-mouse IgG-HRP (Bio-Rad).

Techniques: Infection, Binding Assay, Mutagenesis, Virus, Staining, Standard Deviation, Amplification, Ligation

Journal: bioRxiv

Article Title: A new monoclonal antibody enables BAR analysis of subcellular importin β1 interactomes

doi: 10.1101/2022.03.23.485495

Figure Lengend Snippet: (A) Nucleocytoplasmic transport by importin β1 in complex with an importin α traffics NLS bearing cargos through the nuclear pore complex (NPC). (B) Importin β1 mediated transport in neuronal cytoplasm occurs upon local translation of anterogradely transported importin β1 mRNA, followed by formation of a retrogradely transported importins-cargo complex. K, kinesin; Nucl, nucleolin; D, dynein, β, importin β; α, importin α.

Article Snippet: The antibody was raised against full-length recombinant mouse importin β1 protein (UniProt accession # P70168 ) with an N-terminal His-tag, produced in yeast (MyBioSource: cat # MBS955236).

Techniques:

Journal: bioRxiv

Article Title: A new monoclonal antibody enables BAR analysis of subcellular importin β1 interactomes

doi: 10.1101/2022.03.23.485495

Figure Lengend Snippet: (A) Schematic of the immunization timeline. Full-length recombinant mouse importin β1 (KPNB1) protein was used for the immunization via footpad injection. The mouse with the highest immune response (ELISA screen) was used to generate hybridoma cells by fusing spleen cells with myeloma cells. (B) Schematic of the screening procedure and clone selection. Starting with 960 hybridoma cultures, ∼130 clones tested positive for KPNB1 in ELISA and were expanded in 24 well plates. Supernatants were then tested in immunofluorescence (IF) on 3T3 and HeLa cells. The 20 best performing clones underwent further screening and were scored in different applications: linear ELISA, western blot (WB), and IF on DRG neurons. Sub-cloning and isotyping was performed on the top eight clones. Clone #73 was chosen based on total score (suitability for different applications) and its relative specificity for cytoplasmic KPNB1.

Article Snippet: The antibody was raised against full-length recombinant mouse importin β1 protein (UniProt accession # P70168 ) with an N-terminal His-tag, produced in yeast (MyBioSource: cat # MBS955236).

Techniques: Recombinant, Injection, Enzyme-linked Immunosorbent Assay, Selection, Clone Assay, Immunofluorescence, Western Blot, Subcloning

Journal: bioRxiv

Article Title: A new monoclonal antibody enables BAR analysis of subcellular importin β1 interactomes

doi: 10.1101/2022.03.23.485495

Figure Lengend Snippet: (A) Structural model of importins complex with an NLS-bearing cargo protein, nucleoplasmin, from https://pdb101.rcsb.org/motm/85 . IBB, importin β1 Binding domain of importin α. (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. GS, Glycine-Serine linker. Pink shading around BirA* indicates 10nm range of biotinylation. (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.

Article Snippet: The antibody was raised against full-length recombinant mouse importin β1 protein (UniProt accession # P70168 ) with an N-terminal His-tag, produced in yeast (MyBioSource: cat # MBS955236).

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

Journal: bioRxiv

Article Title: A new monoclonal antibody enables BAR analysis of subcellular importin β1 interactomes

doi: 10.1101/2022.03.23.485495

Figure Lengend Snippet: (A) Serum screening of the immunized mice by ELISA for full length KPNB1 reveal Mouse #3 (M3) as the top candidate. (B) KPNB1 Western blot (WB) using serum derived antibodies of M3. (C-E) Screening using cell supernatants shown for selected clones on WB (C), IF, scale bar 20 µm (D), and linear ELISA (E). (F) Summary scores of the best eight clones that were chosen for purification and isotyping. Scores were between 0 (no signal) to 3 (highest signal). Localization of predominantly cytoplasmic or nuclear (C, N) importin β1 is based on IF images. (G) ELISA (full-length mouse KPNB1 with His-tag) using the top eight antibodies after purification. His-tag alone was used as a negative control (neg.).

Article Snippet: The antibody was raised against full-length recombinant mouse importin β1 protein (UniProt accession # P70168 ) with an N-terminal His-tag, produced in yeast (MyBioSource: cat # MBS955236).

Techniques: Enzyme-linked Immunosorbent Assay, Western Blot, Derivative Assay, Clone Assay, Purification, Negative Control

Journal: bioRxiv

Article Title: A new monoclonal antibody enables BAR analysis of subcellular importin β1 interactomes

doi: 10.1101/2022.03.23.485495

Figure Lengend Snippet: (A) Multiple sequence alignment show homology of the identified epitope, comprising the residues 301-320 of importin β1, with color coding according to the physicochemical property of the amino acid by Clustal Omega. (B) PyMOL generated representations of importin β1 (grey) in complex with the N-terminal IBB domain of importin α (1QGK) or Ran (1IBR) (both in blue), with the identified epitope shown in red. Ran complex shown from two angles. (C-D) ELISA data with serial dilutions of either peptide (C) or mAb (D) confirms dose-dependent recognition.

Article Snippet: The antibody was raised against full-length recombinant mouse importin β1 protein (UniProt accession # P70168 ) with an N-terminal His-tag, produced in yeast (MyBioSource: cat # MBS955236).

Techniques: Sequencing, Generated, Enzyme-linked Immunosorbent Assay

Journal: bioRxiv

Article Title: A new monoclonal antibody enables BAR analysis of subcellular importin β1 interactomes

doi: 10.1101/2022.03.23.485495

Figure Lengend Snippet: (A) Confocal images of adult DRG neurons in culture, stained for importin β1 using mAbKPNB1-301-320 (mAbKPNB1, 4.6 nM). Middle panel after pre-incubation with a peptide comprising the epitope sequence (9.5 nM KPNB1 301-320 ), right panel is omitted primary antibody control. Scale bar 20 ¼ m. (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 immuno-gold labeling of importin β1 in cultured mouse DRG neurons using mAbKPNB1-301-320 (mAbKPNB1, 0.23 μg/μl). Scale bars: 100 nm; gold particle diameter: 10 nm, MT: microtubules. (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.

Article Snippet: The antibody was raised against full-length recombinant mouse importin β1 protein (UniProt accession # P70168 ) with an N-terminal His-tag, produced in yeast (MyBioSource: cat # MBS955236).

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

Journal: bioRxiv

Article Title: A new monoclonal antibody enables BAR analysis of subcellular importin β1 interactomes

doi: 10.1101/2022.03.23.485495

Figure Lengend Snippet: Ab (A) PLA detection of importin β1 using 2.7µg/ml mAbKPNB1-301-320 and 0.5µg/ml polyclonal anti-KPNB1 (MBS713065) in adult DRG neurons. Scale bars-full image 20 µm; insert = 10 µm. (B) PLA quantification in axons, Mean ± SEM, n > 3, t-test, **** indicates p < 0.0001.

Article Snippet: The antibody was raised against full-length recombinant mouse importin β1 protein (UniProt accession # P70168 ) with an N-terminal His-tag, produced in yeast (MyBioSource: cat # MBS955236).

Techniques:

Journal: bioRxiv

Article Title: A new monoclonal antibody enables BAR analysis of subcellular importin β1 interactomes

doi: 10.1101/2022.03.23.485495

Figure Lengend Snippet: (A) Immunostaining of cryosections with mAbKPNB1-301-320 (mAbKPNB1, 2.7ng/μl) before and 24 hrs. after sciatic nerve crush. Scale bar 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, Scale bar 5 μm. (D, 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: The antibody was raised against full-length recombinant mouse importin β1 protein (UniProt accession # P70168 ) with an N-terminal His-tag, produced in yeast (MyBioSource: cat # MBS955236).

Techniques: Immunostaining, Staining, Two Tailed Test, Imaging

Journal: bioRxiv

Article Title: A new monoclonal antibody enables BAR analysis of subcellular importin β1 interactomes

doi: 10.1101/2022.03.23.485495

Figure Lengend Snippet: (A) Paraffin cross sections of the sciatic nerve (SN), showing importin β1 (KPNB1) in red and Neurofilament (NFH) in green. Distance: 5 mm proximal to the site of injury, 6hrs. after SN crush compared to naive. Scale bar 5 μm. (B) Quantification of (A), Mean with SEM, n > 450 axons, unpaired two tailed t-test, **** indicates p < 0.0001.

Article Snippet: The antibody was raised against full-length recombinant mouse importin β1 protein (UniProt accession # P70168 ) with an N-terminal His-tag, produced in yeast (MyBioSource: cat # MBS955236).

Techniques: Two Tailed Test

Journal: bioRxiv

Article Title: A new monoclonal antibody enables BAR analysis of subcellular importin β1 interactomes

doi: 10.1101/2022.03.23.485495

Figure Lengend Snippet: (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. (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.

Article Snippet: The antibody was raised against full-length recombinant mouse importin β1 protein (UniProt accession # P70168 ) with an N-terminal His-tag, produced in yeast (MyBioSource: cat # MBS955236).

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

Journal: Journal of Cell Science

Article Title: Dynamic trafficking of STAT5 depends on an unconventional nuclear localization signal

doi: 10.1242/jcs.123042

Figure Lengend Snippet: Fig. 1.

Article Snippet: Short interfering RNAs (siRNA) specific for human importin-α or β1 (Qiagen Inc., Valencia, CA) were transfected with X-tremeGENE siRNA transfection reagent (Roche, Indianapolis, IN).

Techniques: