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