Importin beta/KPNB1 Antibody (845208)

HAF018). A specific band was detected for Importin beta /KPNB1 at approximately 97 kDa (as indicated). This experiment was conducted under reducing conditions and using Immunoblot Buffer Group 1." > HAF018). A specific band was detected for Importin beta /KPNB1 at approximately 97 kDa (as indicated). This experiment was conducted under reducing conditions and using Immunoblot Buffer Group 1." title="Western blot shows lysates of HeLa human cervical epithelial carcinoma cell line, NIH-3T3 mouse embryonic fibroblast cell line, and Rat-2 rat embryonic fibroblast cell line. PVDF membrane was probed with 1 µg/mL of Mouse Anti-Human Importin beta /KPNB1 Monoclonal Antibody (Catalog # MAB8209) followed by HRP-conjugated Anti-Mouse IgG Secondary Antibody (Catalog # HAF018). A specific band was detected for Importin beta /KPNB1 at approximately 97 kDa (as indicated). This experiment was conducted under reducing conditions and using Immunoblot Buffer Group 1." />

Western blot shows lysates of HeLa human cervical epithelial carcinoma cell line, NIH-3T3 mouse embryonic fibroblast cell line, and Rat-2 rat embryonic fibroblast cell line. PVDF membrane was probed with 1 µg/mL of Mouse Anti-Human Importin beta /KPNB1 Monoclonal Antibody (Catalog # MAB8209) followed by HRP-conjugated Anti-Mouse IgG Secondary Antibody (Catalog # <a class=

NL007) and counterstained with DAPI (blue). Specific staining was localized to nuclei. View our protocol for Fluorescent ICC Staining of Cells on Coverslips." > NL007) and counterstained with DAPI (blue). Specific staining was localized to nuclei. View our protocol for Fluorescent ICC Staining of Cells on Coverslips." title="Importin beta /KPNB1 was detected in immersion fixed HeLa human cervical epithelial carcinoma cell line using Mouse Anti-Human Importin beta /KPNB1 Monoclonal Antibody (Catalog # MAB8209) at 25 µg/mL for 3 hours at room temperature. Cells were stained using the NorthernLights™ 557-conjugated Anti-Mouse IgG Secondary Antibody (red; Catalog # NL007) and counterstained with DAPI (blue). Specific staining was localized to nuclei. View our protocol for Fluorescent ICC Staining of Cells on Coverslips." />

Importin beta /KPNB1 was detected in immersion fixed HeLa human cervical epithelial carcinoma cell line using Mouse Anti-Human Importin beta /KPNB1 Monoclonal Antibody (Catalog # MAB8209) at 25 µg/mL for 3 hours at room temperature. Cells were stained using the NorthernLights™ 557-conjugated Anti-Mouse IgG Secondary Antibody (red; Catalog # <a class=

cNLSs are necessary for Gli2 nuclear import.(A) Sequences in Gli2_Mm predicted as cNLSs by cNLS-Mapper [40] 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 Fig 1C. 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-alpha 1 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). Image collected and cropped by CiteAb from the following publication (//pubmed.ncbi.nlm.nih.gov/27579771), licensed under a CC-BY license. Not internally tested by R&D Systems.

Imp-alpha / beta 1 transports Gli2 into the nucleus.(A) Two segments of a T-Coffee [69] alignment of four Gli-like sequences: GLI2_Mm (Mus musculus), GLI2_Hs, GLI1_Hs, GLI3_Hs (Homo sapiens) and Ci_Dm (Drosophila melanogaster). Residue similarity is colour coded according to the Risler substitution matrix, using ESPript [70]. 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 [40] for GLI2_Mm. (B) GFP-Gli2-NIH/3T3 cells were treated with Importazol (IPZ) in order to inhibit Imp-beta 1-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-beta 1 (F) and Imp-alpha 1 (G) after precipitating GFP-Gli2 with GFP-Trap (left) from HEK293FT transfected with

Imp-alpha / beta 1 transports Gli2 into the nucleus.(A) Two segments of a T-Coffee [69] alignment of four Gli-like sequences: GLI2_Mm (Mus musculus), GLI2_Hs, GLI1_Hs, GLI3_Hs (Homo sapiens) and Ci_Dm (Drosophila melanogaster). Residue similarity is colour coded according to the Risler substitution matrix, using ESPript [70]. 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 [40] for GLI2_Mm. (B) GFP-Gli2-NIH/3T3 cells were treated with Importazol (IPZ) in order to inhibit Imp-beta 1-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-beta 1 (F) and Imp-alpha 1 (G) after precipitating GFP-Gli2 with GFP-Trap (left) from HEK293FT transfected with

Imp-beta 2 is involved in moving Gli2 into the cilium.(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-beta 2 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-beta 2 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 Fig 1C. 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-bas

• Reactivity: Hu, Mu, Rt
• Applications: WB, ICC/IF
• Clone: 845208
• Clonality: Monoclonal
• Host: Mouse
• Conjugate: Unconjugated
• Concentration: LYOPH

Importin beta/KPNB1 Antibody (845208) Summary

• Immunogen: E. coli-derived recombinant human Importin beta /KPNB1
  Met1-Gly155
  Accession # Q14974
• Specificity: Detects human Importin beta /KPNB1 in ELISA and Western Blot. It detects mouse and rat Importin beta /KPNB1 in Western Blots.
• Source: N/A
• Isotype: IgG2b
• Clonality: Monoclonal
• Host: Mouse
• Gene: KPNB1
• Purity Statement: Protein A or G purified from hybridoma culture supernatant

Applications/Dilutions

• Dilutions:
  • Immunocytochemistry 8-25 ug/mL
  • Western Blot 1 ug/mL

Packaging, Storage & Formulations

• Storage: Use a manual defrost freezer and avoid repeated freeze-thaw cycles.
  • 12 months from date of receipt, -20 to -70 °C as supplied.
  • 1 month, 2 to 8 °C under sterile conditions after reconstitution.
  • 6 months, -20 to -70 °C under sterile conditions after reconstitution.
• Buffer: Lyophilized from a 0.2 μm filtered solution in PBS with Trehalose. *Small pack size (SP) is supplied either lyophilized or as a 0.2 µm filtered solution in PBS.
• Preservative: No Preservative
• Concentration: LYOPH
• Reconstitution Instructions: Reconstitute at 0.5 mg/mL in sterile PBS.

Notes

This product is produced by and ships from R&D Systems, Inc., a Bio-Techne brand.

Alternate Names for Importin beta/KPNB1 Antibody (845208)

• IMB1
• Impnb
• Importin 1
• importin 90
• Importin beta
• importin beta-1 subunit
• importin subunit beta-1
• importin-90
• IPO1
• IPOB
• karyopherin (importin) beta 1
• Karyopherin subunit beta-1
• KPNB1
• MGC2155
• MGC2156
• MGC2157
• NTF97
• NTF97Pore targeting complex 97 kDa subunit
• Nuclear factor p97
• PTAC97

Background

KPNB1 (Karyopherin subunit beta 1), also called Importin beta 1, NTF97 (nuclear transport factor, 97 kDa) or PTAC97 (pore targeting complex, 97 kDa), is a member of the Importin b family of proteins. It is ubiquitously expressed and plays many roles in the nucleus. Primarily, KPNB1 transports various complexes from cytoplasm into nucleus, either alone or with a KPNA adaptor. The 876 amino acid (aa) human KPNB1 contains an N-terminal IBB/Importin b domain (aa 21-101), and eight HEAT (solenoid) domains (aa 124-726). A 731 aa splice variant begins with Met146, while an 845 aa variant shows a 2 aa substitution for aa 702-734. Binding sites for adaptors and for its regulator, Ran-GTP, overlap within the HEAT domains. Human KPNB1 aa 1-155 shares 99% and 97% aa sequence identity with mouse and rat KPNB1, respectively.

Limitations

This product is for research use only and is not approved for use in humans or in clinical diagnosis. Primary Antibodies are guaranteed for 1 year from date of receipt.

Publications for Importin beta/KPNB1 Antibody (MAB8209)(2)

Publications using MAB8209

• Fan Yang, Lin Li, Zhenzhen Mu, Pengyue Liu, Ying Wang, Yue Zhang et al. Tumor-promoting properties of karyopherin beta 1 in melanoma by stabilizing Ras-GTPase-activating protein SH3 domain-binding protein 1 Cancer Gene Therapy 12/1/2022 [PMID: 35902727]
• Ciliary Entry of the Hedgehog Transcriptional Activator Gli2 Is Mediated by the Nuclear Import Machinery but Differs from Nuclear Transport in Being Imp-?/?1-Independent PLoS ONE, 2016-08-31;11(8):e0162033. 2016-08-31 [PMID: 27579771] (Western Blot, Mouse)

Bioinformatics

• Gene Symbol: KPNB1
• Uniprot:
  • Q14974(Human)