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![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. *p0.05 (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-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 Fig 1C. Results are representative of two experiments and at least 50 cells were analysed for each condition.* p<0.05, *** p<0.0001 (ANOVA). 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.](http://images.novusbio.com/fullsize/mab8209_human-mouse-rat-importin-beta-kpnb1-mab-clone-845208-41202412521121.jpg)
![Immunohistochemistry-Paraffin: Importin beta/KPNB1 Antibody [NBP1-87712] - Staining in human testis and skeletal muscle tissues . Corresponding KPNB1 RNA-seq data are presented for the same tissues.](http://images.novusbio.com/fullsize/Importin-beta-KPNB1-Antibody-Immunohistochemistry-Paraffin-NBP1-87712-img0020.jpg "<b>Orthogonal Strategies Validation. </b>Immunohistochemistry-Paraffin: Importin beta/KPNB1 Antibody [NBP1-87712] - Staining in human testis and skeletal muscle tissues . Corresponding KPNB1 RNA-seq data are presented for the same tissues.")
![<b>Orthogonal Strategies Validation. </b>Western Blot: Importin beta/KPNB1 Antibody [NBP1-87712] - Analysis in mouse cell line NIH-3T3 and rat cell line NBT-II.](http://images.novusbio.com/fullsize/Importin-beta-KPNB1-Antibody-Western-Blot-NBP1-87712-img0008.jpg)
![Immunocytochemistry/Immunofluorescence: Importin beta/KPNB1 Antibody [NBP1-87712] - Staining of human cell line A-431 shows localization to nucleoplasm, nuclear membrane & cytosol. Antibody staining is shown in green.](http://images.novusbio.com/fullsize/Importin-beta-KPNB1-Antibody-Immunocytochemistry-Immunofluorescence-NBP1-87712-img0010.jpg)
![Western Blot: Importin beta/KPNB1 Antibody [NBP1-87712] - Analysis in human cell line A-549.](http://images.novusbio.com/fullsize/Importin-beta-KPNB1-Antibody-Western-Blot-NBP1-87712-img0007.jpg)
![Immunohistochemistry-Paraffin: Importin beta/KPNB1 Antibody [NBP1-87712] - Staining of human cerebral cortex shows moderate to strong nuclear and nuclear membrane positivity in neurons.](http://images.novusbio.com/fullsize/Importin-beta-KPNB1-Antibody-Immunohistochemistry-Paraffin-NBP1-87712-img0015.jpg)
![Immunohistochemistry-Paraffin: Importin beta/KPNB1 Antibody [NBP1-87712] - Staining of human skeletal muscle shows very weak nuclear positivity in myocytes.](http://images.novusbio.com/fullsize/Importin-beta-KPNB1-Antibody-Immunohistochemistry-Paraffin-NBP1-87712-img0017.jpg)
![Immunohistochemistry-Paraffin: Importin beta/KPNB1 Antibody [NBP1-87712] - Staining of human stomach shows moderate nuclear and nuclear membrane positivity in glandular cells.](http://images.novusbio.com/fullsize/Importin-beta-KPNB1-Antibody-Immunohistochemistry-Paraffin-NBP1-87712-img0018.jpg)
![Immunohistochemistry-Paraffin: Importin beta/KPNB1 Antibody [NBP1-87712] - Staining of human testis shows moderate to strong nuclear and nuclear membrane positivity in cells in seminiferous ducts.](http://images.novusbio.com/fullsize/Importin-beta-KPNB1-Antibody-Immunohistochemistry-Paraffin-NBP1-87712-img0019.jpg)
![Western Blot: Importin beta/KPNB1 Antibody [NB100-40784] - Staining of A431 (A), HEK293 (B), Jurkat (C) cell lysates with antibody at 0.03 ug/mL, Daudi (D), HeLa (E) cell lysates with antibody at 0.01 ug/mL, and Kelly (F) and KNRK (G) cell lysate with antibody at 0.1 ug/mL (35 ug protein in RIPA buffer). Detected by chemiluminescence.](http://images.novusbio.com/fullsize/Importin-beta-KPNB1-Antibody-Western-Blot-NB100-40784-img0001.jpg "Western Blot: Importin beta/KPNB1 Antibody [NB100-40784] - Staining of A431 (A), HEK293 (B), Jurkat (C) cell lysates with antibody at 0.03 ug/mL, Daudi (D), HeLa (E) cell lysates with antibody at 0.01 ug/mL, and Kelly (F) and KNRK (G) cell lysate with antibody at 0.1 ug/mL (35 ug protein in RIPA buffer). Detected by chemiluminescence.")
![Immunocytochemistry/Immunofluorescence: Importin beta/KPNB1 Antibody [NB100-40784] - Immunofluorescence analysis of paraformaldehyde fixed U2OS cells, permeabilized with 0.15% Triton. Primary incubation 1hr (10 ug/mL) followed by Alexa Fluor 488 secondary antibody (2 ug/mL), showing nuclear membrane, nuclear and cytoplasmic staining. The nuclear stain is DAPI (blue). Negative control: Unimmunized goat IgG (10 ug/mL) followed by Alexa Fluor 488 secondary antibody (2 ug/mL).](http://images.novusbio.com/fullsize/Importin-beta-KPNB1-Antibody-Immunocytochemistry-Immunofluorescence-NB100-40784-img0002.jpg)
![Immunohistochemistry-Paraffin: Importin beta/KPNB1 Antibody [NB100-40784] - Staining of paraffin embedded Human Spleen with antibody at 5 ug/mL. Steamed antigen retrieval with citrate buffer pH 6, AP-staining.](http://images.novusbio.com/fullsize/Importin-beta-KPNB1-Antibody-Immunohistochemistry-Paraffin-NB100-40784-img0006.jpg)
![Flow Cytometry: Importin beta/KPNB1 Antibody [NB100-40784] - Flow cytometric analysis of paraformaldehyde fixed A431 cells (blue line), permeabilized with 0.5% Triton. Primary incubation 1hr (10 ug/mL) followed by Alexa Fluor 488 secondary antibody (1 ug/mL). IgG control: Unimmunized goat IgG (black line) followed by Alexa Fluor 488 secondary antibody.](http://images.novusbio.com/fullsize/Importin-beta-KPNB1-Antibody-Flow-Cytometry-NB100-40784-img0003.jpg)
![Immunoprecipitation: Importin beta/KPNB1 Antibody (PSH01-60) [NBP3-32511] - Importin beta/KPNB1 was immunoprecipitated in 0.2mg HeLa cell lysate with NBP3-32511 at 2 ug/25 ul agarose. Western blot was performed from the immunoprecipitate using NBP3-32511 at 1/1,000 dilution. Anti-Rabbit IgG for IP Nano-secondary antibody at 1/5,000 dilution was used for 1 hour at room temperature. Lane 1: HeLa cell lysate (input) Lane 2: Rabbit IgG instead of NBP3-32511 in HeLa cell lysate Lane 3: NBP3-32511 IP in HeLa cell lysate Blocking/Dilution buffer: 5% NFDM/TBST Exposure time: 24 seconds](http://images.novusbio.com/fullsize/nbp3-32511_rabbit-importin-beta-kpnb1-mab-psh01-60-2682024158263.jpg "Immunoprecipitation: Importin beta/KPNB1 Antibody (PSH01-60) [NBP3-32511] - Importin beta/KPNB1 was immunoprecipitated in 0.2mg HeLa cell lysate with NBP3-32511 at 2 ug/25 ul agarose. Western blot was performed from the immunoprecipitate using NBP3-32511 at 1/1,000 dilution. Anti-Rabbit IgG for IP Nano-secondary antibody at 1/5,000 dilution was used for 1 hour at room temperature. Lane 1: HeLa cell lysate (input) Lane 2: Rabbit IgG instead of NBP3-32511 in HeLa cell lysate Lane 3: NBP3-32511 IP in HeLa cell lysate Blocking/Dilution buffer: 5% NFDM/TBST Exposure time: 24 seconds")
![Western Blot: Importin beta/KPNB1 Antibody (PSH01-60) [NBP3-32511] - Western blot analysis of Importin beta/KPNB1 on different lysates with Rabbit anti-Importin beta/KPNB1 antibody (NBP3-32511) at 1/1,000 dilution. Lane 1: HeLa cell lysate (20 ug/Lane) Lane 2: HepG2 cell lysate (20 ug/Lane) Lane 3: A431 cell lysate (20 ug/Lane) Lane 4: A549 cell lysate (20 ug/Lane) Lane 5: Jurkat cell lysate (20 ug/Lane) Lane 6: K-562 cell lysate (20 ug/Lane) Lane 7: Caco-2 cell lysate (20 ug/Lane) Lane 8: NIH/3T3 cell lysate (20 ug/Lane) Lane 9: PC-12 cell lysate (20 ug/Lane) Lane 10: C6 cell lysate (20 ug/Lane) Lane 11: Mouse spleen tissue lysate (40 ug/Lane) Lane 12: Mouse testis tissue lysate (40 ug/Lane) Lane 13: Rat spleen tissue lysate (40 ug/Lane) Lane 14: Rat testis tissue lysate (40 ug/Lane) Predicted band size: 97 kDa Observed band size: 97 kDa Exposure time: 1 minute; 4-20% SDS-PAGE gel. Proteins were transferred to a PVDF membrane and blocked with 5% NFDM/TBST for 1 hour at room temperature. The primary antibody (NBP3-32511) at 1/1,000 dilution was used in 5% NFDM/TBST at room temperature for 2 hours. Goat Anti-Rabbit IgG - HRP Secondary Antibody at 1/100,000 dilution was used for 1 hour at room temperature.](http://images.novusbio.com/fullsize/nbp3-32511_rabbit-importin-beta-kpnb1-mab-psh01-60-27820241628523.jpg)
![Immunohistochemistry: Importin beta/KPNB1 Antibody (PSH01-60) [NBP3-32511] - Immunofluorescence analysis of paraffin-embedded human testis tissue labeling Importin beta/KPNB1 with Rabbit anti-Importin beta/KPNB1 antibody (NBP3-32511) at 1/200 dilution. The section was pre-treated using heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0) for 20 minutes. The tissues were blocked in 10% negative goat serum for 1 hour at room temperature, washed with PBS, and then probed with the primary antibody (NBP3-32511, green) at 1/200 dilution overnight at 4 ℃, washed with PBS. Goat Anti-Rabbit IgG H&L (iFluor™ 488) was used as the secondary antibody at 1/1,000 dilution. Nuclei were counterstained with DAPI (blue).](http://images.novusbio.com/fullsize/nbp3-32511_rabbit-importin-beta-kpnb1-mab-psh01-60-29820241420963.jpg)
![Immunohistochemistry: Importin beta/KPNB1 Antibody (PSH01-60) [NBP3-32511] - Immunohistochemical analysis of paraffin-embedded human colon tissue with Rabbit anti-Importin beta/KPNB1 antibody (NBP3-32511) at 1/1,000 dilution. The section was pre-treated using heat mediated antigen retrieval with sodium citrate buffer (pH 6.0) for 2 minutes. The tissues were blocked in 1% BSA for 20 minutes at room temperature, washed with ddH2O and PBS, and then probed with the primary antibody (NBP3-32511) at 1/1,000 dilution for 1 hour at room temperature. The detection was performed using an HRP conjugated compact polymer system. DAB was used as the chromogen. Tissues were counterstained with hematoxylin and mounted with DPX.](http://images.novusbio.com/fullsize/nbp3-32511_rabbit-importin-beta-kpnb1-mab-psh01-60-29820241435256.jpg)
![Western Blot: Importin beta/KPNB1 Antibody [NB100-94993] - Detection of Human and Mouse KPNB1 on HeLa whole cell lysate using NB100-94993.](http://images.novusbio.com/fullsize/Importin-beta-KPNB1-Antibody-Western-Blot-NB100-94993-img0001.jpg "Western Blot: Importin beta/KPNB1 Antibody [NB100-94993] - Detection of Human and Mouse KPNB1 on HeLa whole cell lysate using NB100-94993.")
![Western Blot: Importin beta/KPNB1 Antibody [NB100-79806] - Importin beta Antibody [NB100-79806] - Detection of KapB1 by Western Blot and Immunoprecipitation. Samples: A. Whole cell lysate (50 mcg) from HeLa, 293T and mouse 3T3 cells. B. Whole cell lysate (1 mg for IP, 1/4 of IP loaded/lane) from HeLa cells. Antibodies: Affinity purified rabbit anti-KapB1 antibody BL1964 (Cat. No. NB100-79806) used at 0.1 mcg/ml for WB (A and B) and at 3 mcg/mg lysate for IP (B). Immunoprecipitation of KapB1 was unsuccessful when using rabbit anti-KapB1 antibody BL1962 (Cat. No. NB100-79805) at 3 mcg/mg lysate. Detection: Chemiluminescence with an exposure time of 30 seconds (A and B).](http://images.novusbio.com/fullsize/Importin-beta-KPNB1-Antibody-Western-Blot-NB100-79806-img0002.jpg "Western Blot: Importin beta/KPNB1 Antibody [NB100-79806] - Importin beta Antibody [NB100-79806] - Detection of KapB1 by Western Blot and Immunoprecipitation. Samples: A. Whole cell lysate (50 mcg) from HeLa, 293T and mouse 3T3 cells. B. Whole cell lysate (1 mg for IP, 1/4 of IP loaded/lane) from HeLa cells. Antibodies: Affinity purified rabbit anti-KapB1 antibody BL1964 (Cat. No. NB100-79806) used at 0.1 mcg/ml for WB (A and B) and at 3 mcg/mg lysate for IP (B). Immunoprecipitation of KapB1 was unsuccessful when using rabbit anti-KapB1 antibody BL1962 (Cat. No. NB100-79805) at 3 mcg/mg lysate. Detection: Chemiluminescence with an exposure time of 30 seconds (A and B).")
![Immunoprecipitation: Importin beta/KPNB1 Antibody (PSH01-59) [NBP3-32510] - Importin beta/KPNB1 was immunoprecipitated in 0.2mg HeLa cell lysate with NBP3-32510 at 2 ug/25 ul agarose. Western blot was performed from the immunoprecipitate using NBP3-32510 at 1/1,000 dilution. Anti-Rabbit IgG for IP Nano-secondary antibody at 1/5,000 dilution was used for 1 hour at room temperature. Lane 1: HeLa cell lysate (input) Lane 2: Rabbit IgG instead of NBP3-32510 in HeLa cell lysate Lane 3: NBP3-32510 IP in HeLa cell lysate Blocking/Dilution buffer: 5% NFDM/TBST Exposure time: 24 seconds](http://images.novusbio.com/fullsize/nbp3-32510_rabbit-importin-beta-kpnb1-mab-psh01-59-2682024158260.jpg "Immunoprecipitation: Importin beta/KPNB1 Antibody (PSH01-59) [NBP3-32510] - Importin beta/KPNB1 was immunoprecipitated in 0.2mg HeLa cell lysate with NBP3-32510 at 2 ug/25 ul agarose. Western blot was performed from the immunoprecipitate using NBP3-32510 at 1/1,000 dilution. Anti-Rabbit IgG for IP Nano-secondary antibody at 1/5,000 dilution was used for 1 hour at room temperature. Lane 1: HeLa cell lysate (input) Lane 2: Rabbit IgG instead of NBP3-32510 in HeLa cell lysate Lane 3: NBP3-32510 IP in HeLa cell lysate Blocking/Dilution buffer: 5% NFDM/TBST Exposure time: 24 seconds")
![Western Blot: Importin beta/KPNB1 Antibody (PSH01-59) [NBP3-32510] - Western blot analysis of Importin beta/KPNB1 on different lysates with Rabbit anti-Importin beta/KPNB1 antibody (NBP3-32510) at 1/1,000 dilution. Lane 1: HeLa cell lysate (20 ug/Lane) Lane 2: HepG2 cell lysate (20 ug/Lane) Lane 3: A431 cell lysate (20 ug/Lane) Lane 4: A549 cell lysate (20 ug/Lane) Lane 5: Jurkat cell lysate (20 ug/Lane) Lane 6: K-562 cell lysate (20 ug/Lane) Lane 7: Caco-2 cell lysate (20 ug/Lane) Lane 8: NIH/3T3 cell lysate (20 ug/Lane) Lane 9: PC-12 cell lysate (20 ug/Lane) Lane 10: C6 cell lysate (20 ug/Lane) Lane 11: Mouse spleen tissue lysate (40 ug/Lane) Lane 12: Mouse testis tissue lysate (40 ug/Lane) Lane 13: Rat spleen tissue lysate (40 ug/Lane) Lane 14: Rat testis tissue lysate (40 ug/Lane) Predicted band size: 97 kDa Observed band size: 97 kDa Exposure time: 1 minute; 4-20% SDS-PAGE gel. Proteins were transferred to a PVDF membrane and blocked with 5% NFDM/TBST for 1 hour at room temperature. The primary antibody (NBP3-32510) at 1/1,000 dilution was used in 5% NFDM/TBST at room temperature for 2 hours. Goat Anti-Rabbit IgG - HRP Secondary Antibody at 1/100,000 dilution was used for 1 hour at room temperature.](http://images.novusbio.com/fullsize/nbp3-32510_rabbit-importin-beta-kpnb1-mab-psh01-59-278202415295597.jpg)
![Immunocytochemistry/Immunofluorescence: Importin beta/KPNB1 Antibody [NBP2-55641] - Staining of human cell line A-431 shows localization to nucleoplasm, nuclear membrane & cytosol.](http://images.novusbio.com/fullsize/Importin-beta-KPNB1-Antibody-Immunocytochemistry-Immunofluorescence-NBP2-55641-img0001.jpg "Immunocytochemistry/Immunofluorescence: Importin beta/KPNB1 Antibody [NBP2-55641] - Staining of human cell line A-431 shows localization to nucleoplasm, nuclear membrane & cytosol.")
![SDS-Page: Recombinant Human Importin beta/KPNB1 Protein [NBP1-78815] - 3ug by SDS-PAGE under reducing condition and visualized by coomassie blue stain.](http://images.novusbio.com/fullsize/Recombinant-Human-Importin-beta-KPNB1-Protein-SDS-Page-NBP1-78815-img0001.jpg "SDS-Page: Recombinant Human Importin beta/KPNB1 Protein [NBP1-78815] - 3ug by SDS-PAGE under reducing condition and visualized by coomassie blue stain.")