Journal: Frontiers in Oncology

Article Title: YTHDF1 Aggravates the Progression of Cervical Cancer Through m 6 A-Mediated Up-Regulation of RANBP2

doi: 10.3389/fonc.2021.650383

Figure Lengend Snippet: RANBP2 is the key target of YTHDF1 in cervical cancer. (A) Western blot detecting the protein level of RANBP2 in Hela and Siha cells upon YTHDF1 knockdown. (B) RT-qPCR detecting relative RNA level of RANBP2 in Hela and Siha upon YTHDF1 knockdown. (C) RIP-PCR assays detecting the interactions between YTHDF1 and RANBP2 mRNA in Siha cells. IgG was used as an internal control. GAPDH was used as the negative control in western blot assays. (D) meRIP-PCR assays detecting the m 6 A modification of RANBP2 mRNA in Siha cells. (E) Schematic of wild-type (YTHDF1-wt) and mutant (YTHDF1-mut) YTHDF1 constructs. (F) RIP-derived RNA and protein of wild-type (YTHDF1-wt) group and mutant (YTHDF1-mut) group in Hela cells were measured by RT-qPCR and western blot after immunoprecipitation by using the antibody specific to Flag, respectively. GAPDH was used as the negative control in western blot assays. Data are shown as means ± S.D. **P < 0.01, ***P < 0.001.

Article Snippet: The slices were washed three times with PBS, and blocked with goat serum (ZSGB-BIO, ZLI-9021) at 37°C for 30 min. Rabbit anti-YTHDF1 antibody (ProteinTech, 1:100) or anti-RANBP2 antibody (ProteinTech, 1:100) was added to the slices and incubated overnight at 4°C.

Techniques: Western Blot, Knockdown, Quantitative RT-PCR, Control, Negative Control, Modification, Mutagenesis, Construct, Derivative Assay, Immunoprecipitation

Journal: Frontiers in Oncology

Article Title: YTHDF1 Aggravates the Progression of Cervical Cancer Through m 6 A-Mediated Up-Regulation of RANBP2

doi: 10.3389/fonc.2021.650383

Figure Lengend Snippet: RANBP2 plays an oncogenic role in cervical cancer cells. (A) Detection of RANBP2 knockdown in Hela and Siha cell lines by western blot. (B) The effect of RANBP2 knockdown on cell growth was determined by CCK-8 assays. (C) Colony formation assays were performed in RANBP2 knockdown and control cells. (D, E) Migration and invasion assays of Hela and Siha cells upon RANBP2 knockdown. Scale bar, 200 μm. Data are shown as means ± S.D. **P < 0.01, ***P < 0.001.

Article Snippet: The slices were washed three times with PBS, and blocked with goat serum (ZSGB-BIO, ZLI-9021) at 37°C for 30 min. Rabbit anti-YTHDF1 antibody (ProteinTech, 1:100) or anti-RANBP2 antibody (ProteinTech, 1:100) was added to the slices and incubated overnight at 4°C.

Techniques: Knockdown, Western Blot, CCK-8 Assay, Control, Migration

Journal: Frontiers in Oncology

Article Title: YTHDF1 Aggravates the Progression of Cervical Cancer Through m 6 A-Mediated Up-Regulation of RANBP2

doi: 10.3389/fonc.2021.650383

Figure Lengend Snippet: Knockdown of RANBP2 suppressed the proliferation, migration and invasion of YTHDF1-overexpressing Hela and Siha cells. (A) Colony formation assays were performed in YTHDF1-overexpressing Hela and Siha cells infected with the RANBP2 shRNA or controls. (B) The proliferation ability of YTHDF1-overexpressing Hela and Siha cells upon RANBP2 knockdown was assessed by CCK-8 assays. (C, D) Migration and invasion YTHDF1-overexpressing Hela (C) and Siha (D) cells upon RANBP2 knockdown was detected by transwell assays. Scale bar, 200 μm. *P < 0.05,**P < 0.01, ***P < 0.001.

Article Snippet: The slices were washed three times with PBS, and blocked with goat serum (ZSGB-BIO, ZLI-9021) at 37°C for 30 min. Rabbit anti-YTHDF1 antibody (ProteinTech, 1:100) or anti-RANBP2 antibody (ProteinTech, 1:100) was added to the slices and incubated overnight at 4°C.

Techniques: Knockdown, Migration, Infection, shRNA, CCK-8 Assay

Journal: Frontiers in Oncology

Article Title: YTHDF1 Aggravates the Progression of Cervical Cancer Through m 6 A-Mediated Up-Regulation of RANBP2

doi: 10.3389/fonc.2021.650383

Figure Lengend Snippet: The expression of RANBP2 is positively correlated with YTHDF1 in cervical cancer. (A) Representative immunohistochemical images of RANBP2 protein expression in cervical cancer tissues and cervical epithelium tissues. Scale bar, 100 μm. (B) The quantitative analysis of RANBP2 expression in cervical cancer tissues and cervical epithelium tissues assessed by immunohistochemistry. (C) Spearman’s correlation analysis of RANBP2 and YTHDF1 expression in cervical cancer tissues. (D) Representative immunohistochemical images of YTHDF1 and RANBP2 in the cervical cancer tissues. Scale bar, 100 μm. Data are shown as means ± S.D. *P < 0.05.

Article Snippet: The slices were washed three times with PBS, and blocked with goat serum (ZSGB-BIO, ZLI-9021) at 37°C for 30 min. Rabbit anti-YTHDF1 antibody (ProteinTech, 1:100) or anti-RANBP2 antibody (ProteinTech, 1:100) was added to the slices and incubated overnight at 4°C.

Techniques: Expressing, Immunohistochemical staining, Immunohistochemistry

Journal: The Journal of Cell Biology

Article Title: The nucleoporin RanBP2 tethers the cAMP effector Epac1 and inhibits its catalytic activity

doi: 10.1083/jcb.201011126

Figure Lengend Snippet: Epac1 directly interacts with the ZNF domain of RanBP2. (A) Domain architecture of RanBP2 with the fragments of RanBP2 isolated in a yeast two-hybrid screen using full-length Epac1 as bait shown below. RBD, Ran-binding domain; ZNF, zinc finger; IR, internal repeat domain; CHD, cyclophilin homology domain. (B) Coimmunoprecipitation of endogenous RanBP2 with HA-tagged Epac1 in HEK293T cells. Note that besides a major band of 358 kD (asterisks in B–D), multiple additional bands of RanBP2 are present on a 6% SDS-PAGE gel, which all disappear upon siRNA-mediated depletion of RanBP2 ( Fig. S1 B ). (C) Coimmunoprecipitation of endogenous RanBP2 with Epac1, which was immunoprecipitated with a rabbit polyclonal Epac1 antibody but not with preimmune serum (N.I., nonimmune) in Ovcar3 cells. (D) Coimmunoprecipitation of endogenous RanBP2 with HA-tagged Epac1 but not with HA-tagged Epac2 in HEK293T cells. (E) Coimmunoprecipitation of the YFP-tagged ZNF domain of RanBP2 with Flag-tagged Epac1 in HEK293T cells. (F) Coimmunoprecipitation of a YFP-tagged version of one on the individual ZNFs (ZNF #2) of RanBP2 with HA-tagged Epac1 in HEK293T cells. (G) Pull-down of bacterially purified Epac1 with the GST-tagged bacterially purified ZNF domain of RanBP2 and, conversely, the ZNF domain with Epac1. Proteins were visualized by simply blue staining. EV, empty vector; IB, immunoblot; IP, immunoprecipitation; TL, total lysate.

Article Snippet: The following antibodies were used: mouse monoclonal GFP (Roche), Flag M2 (Sigma-Aldrich), rabbit polyclonal RanBP2 (Abcam), Rap1 (Santa Cruz Biotechnology, Inc.), and Ran (BD).

Techniques: Isolation, Two Hybrid Screening, Binding Assay, SDS Page, Immunoprecipitation, Purification, Staining, Plasmid Preparation, Western Blot

Journal: The Journal of Cell Biology

Article Title: The nucleoporin RanBP2 tethers the cAMP effector Epac1 and inhibits its catalytic activity

doi: 10.1083/jcb.201011126

Figure Lengend Snippet: RanBP2 recruits Epac1 to the NPC. (A) Live imaging of YFP-Epac1 together with the nuclear marker H2B-RFP in HEK293T cells showing the localization of Epac1 in the cytosol, the nucleus, and at the nuclear envelope. (B–D) Localization of YFP-Epac1 in HEK293T cells (B) and U2OS cells (C) and of endogenous Epac1 in Ovcar3 cells (D). Cells were transfected with either control (scr, scrambled) or RanBP2 siRNAs, and the HEK293T and U2OS cells were transfected the next day with YFP-Epac1. 60 h after siRNA transfection, cells were fixed and stained for endogenous RanBP2 with the goat polyclonal RanBP2 antibody and also for endogenous Epac1 in the Ovcar3 cells. Epac1 colocalizes with RanBP2 at the nuclear envelope, which is dependent on the presence of the RanBP2. (E) Live imaging of CFP-Epac1 together with YFP–empty vector–CAAX or YFP-ZNF-CAAX in HEK293T cells. Targeting of the ZNFs of RanBP2 to the plasma membrane by addition of the CAAX motif of K-Ras recruits Epac1 to the plasma membrane as well. Bars, 10 µm.

Article Snippet: The following antibodies were used: mouse monoclonal GFP (Roche), Flag M2 (Sigma-Aldrich), rabbit polyclonal RanBP2 (Abcam), Rap1 (Santa Cruz Biotechnology, Inc.), and Ran (BD).

Techniques: Imaging, Marker, Transfection, Control, Staining, Plasmid Preparation, Clinical Proteomics, Membrane

Journal: The Journal of Cell Biology

Article Title: The nucleoporin RanBP2 tethers the cAMP effector Epac1 and inhibits its catalytic activity

doi: 10.1083/jcb.201011126

Figure Lengend Snippet: Epac1 remains bound to RanBP2 at the NPC upon cAMP binding. (A) Coimmunoprecipitation of endogenous RanBP2 with Flag-tagged Epac1 in HEK293T cells after stimulation with 100 µM 8-pCPT-2′- O -Me-cAMP (007) for the indicated time points. (B) Confocal live imaging of HEK293T cells transfected with YFP-Epac1 before and 10 min after stimulation with 1 µM 8-pCPT-2′- O -Me-cAMP-AM (007-AM). Although the cytosolic fraction of Epac1 relocalizes to the plasma membrane upon 007-AM stimulation, its localization at the nuclear envelope remains unaltered. Bar, 5 µm. IB, immunoblot; IP, immunoprecipitation; TL, total lysate.

Article Snippet: The following antibodies were used: mouse monoclonal GFP (Roche), Flag M2 (Sigma-Aldrich), rabbit polyclonal RanBP2 (Abcam), Rap1 (Santa Cruz Biotechnology, Inc.), and Ran (BD).

Techniques: Binding Assay, Imaging, Transfection, Clinical Proteomics, Membrane, Western Blot, Immunoprecipitation

Journal: The Journal of Cell Biology

Article Title: The nucleoporin RanBP2 tethers the cAMP effector Epac1 and inhibits its catalytic activity

doi: 10.1083/jcb.201011126

Figure Lengend Snippet: Phosphorylation of the ZNFs of RanBP2 releases Epac1 from the NPC. (A) Coimmunoprecipitation of endogenous RanBP2 with Flag-tagged Epac1 in U2OS cells that were arrested in mitosis by 24-h incubation with 2.5 µM thymidine, washed three times with PBS, and incubated for 16 h with 250 ng/ml nocodazole. Mitotic cells were subsequently collected by mitotic shake off and subjected to coimmunoprecipitation. (B) Coimmunoprecipitation of endogenous RanBP2 with Flag-tagged Epac1 in U2OS cells that were arrested in mitosis similar to panel A but with treatment of the lysate with 1 µM λ-phosphatase for 30 min before immunoprecipitation of Flag-Epac1. The quantification shows the mean with standard deviation of the relative binding of RanBP2 to Flag-Epac1 from three independent experiments. Statistical analysis was performed using a one-tailed Student’s t test. Asterisks indicate the p-value of the respective sample with the associated control sample. *, P < 0.007; **, P < 0.0005. (C) Coimmunoprecipitation of the YFP-tagged individual ZNF (ZNF #2) of RanBP2 (YFP-ZNF) with Epac1 in U2OS cells that were arrested in mitosis similar to panel A. (D) Coimmunoprecipitation of the YFP-tagged individual ZNF (ZNF #2) of RanBP2 (YFP-ZNF) with Flag-tagged Epac1 in HEK293T cells after stimulation with the phosphatase inhibitor okadaic acid (OA; 1 µM) for 1 h. OA results in a decrease in electrophoretic mobility of both YFP-ZNF and Flag-Epac1 and a decrease in association of the two proteins. (E) Confocal live imaging of HEK293T cells transfected with YFP-Epac1 before and 1 h after stimulation with 1 µM OA. This shows the decreased presence of Epac1 at the nuclear envelope upon OA stimulation. Note that also the plasma membrane localization of Epac1 is affected by OA stimulation, whereas an increase in cytosolic YFP-Epac1 is observed. Bar, 2 µm. (F) Coimmunoprecipitation of YFP-ZNF with Flag-tagged Epac1 after selective induction of phosphorylation of either YFP-ZNF or Flag-Epac1 by OA. Two separate dishes of HEK293T cells were transfected with either YFP-ZNF or Flag-Epac1 where indicated, and either one of the two or both were stimulated with 1 µM OA. Subsequently, the cell lysates of YFP-ZNF– and Flag-Epac1–expressing cells were mixed and subjected to coimmunoprecipitation. This demonstrates that OA stimulation of YFP-ZNF–transfected cells, but not Flag-Epac1–transfected cells, inhibits the binding between the two proteins. The quantification shows the mean with standard deviation of the relative binding of YFP-ZNF to Flag-Epac1 from three independent experiments. Statistical analysis was performed using a one-tailed Student’s t test. *, P < 0.0001. The minus signs indicate unstimulated cells. IB, immunoblot; IP, immunoprecipitation; TL, total lysate.

Article Snippet: The following antibodies were used: mouse monoclonal GFP (Roche), Flag M2 (Sigma-Aldrich), rabbit polyclonal RanBP2 (Abcam), Rap1 (Santa Cruz Biotechnology, Inc.), and Ran (BD).

Techniques: Phospho-proteomics, Incubation, Immunoprecipitation, Standard Deviation, Binding Assay, One-tailed Test, Control, Imaging, Transfection, Clinical Proteomics, Membrane, Expressing, Western Blot

Journal: The Journal of Cell Biology

Article Title: The nucleoporin RanBP2 tethers the cAMP effector Epac1 and inhibits its catalytic activity

doi: 10.1083/jcb.201011126

Figure Lengend Snippet: RanBP2 binds to the CDC25-HD of Epac1. (A) Domain architecture of Epac1 showing its catalytic region with the CDC25-homology domain (CDC25-HD) responsible for the catalysis of Rap, which is stabilized by the Ras exchange motif (REM) domain. In addition, the catalytic region contains a Ras association (RA) motif. The regulatory region of Epac1 contains the cAMP-binding (CNB) domain and a Disheveled, Egl-10, and Pleckstrin (DEP) domain. (B) Coimmunoprecipitation of endogenous RanBP2 with YFP-tagged full-length Epac1, the individual regulatory region (Reg Region), catalytic region (Cat Region), and the individual domains from the catalytic region of Epac1 in HEK293T cells. The CDC25-HD of Epac1 mediates the binding to RanBP2. (C) Confocal live imaging of HEK293T cells transfected with the YFP-tagged individual regulatory region, catalytic region, and the individual domains from the catalytic region of Epac1. Both the catalytic region and the CDC25-HD localize to the nuclear envelope. (D) Confocal live imaging of HEK293T cells transfected with YFP-tagged Epac1 lacking its RA domain (YFP-Epac1-ΔRA) together with CFP-tagged wild-type Epac1 showing that Epac1 lacking its RA domain is recruited to the nuclear envelope similar to wild-type Epac1. To maintain structural integrity, the RA domain of Epac1 is replaced by the region linking the REM domain and the CDC25-HD of the RasGEF Sos, which lacks an RA domain. Bars, 10 µm. IB, immunoblot; IP, immunoprecipitation; TL, total lysate.

Article Snippet: The following antibodies were used: mouse monoclonal GFP (Roche), Flag M2 (Sigma-Aldrich), rabbit polyclonal RanBP2 (Abcam), Rap1 (Santa Cruz Biotechnology, Inc.), and Ran (BD).

Techniques: Binding Assay, Imaging, Transfection, Western Blot, Immunoprecipitation

Journal: The Journal of Cell Biology

Article Title: The nucleoporin RanBP2 tethers the cAMP effector Epac1 and inhibits its catalytic activity

doi: 10.1083/jcb.201011126

Figure Lengend Snippet: RanBP2-binding inhibits GEF activity of Epac1. (A) Measurement of Epac1 activity in vitro. Rap1B was loaded with fluorescent mantGDP and the release of mantGDP by Epac1Δ1–148 (left) or Epac2 (right) in the presence of 10 µM cAMP was measured in real time in the absence or presence of 1 or 10 µM of the zinc finger (ZNF) domain of RanBP2 (which, as shown in , targets Epac1 to the NPC in vivo). The presented data are representatives of three independent experiments. (B) Pull-down of Rap1-GTP from Ovcar3 cells upon stimulation for 5 min with 5 µM isoproterenol (iso) 60 h after transfection with control (scr, scrambled), RanBP2, or Epac1 siRNAs. The bottom blot shows the efficiency of the knockdown of RanBP2 and Epac1. The quantification shows the mean with standard deviation of Rap1 activity from four independent experiments. Statistical analysis was performed using a one-tailed Student’s t test. Asterisks indicate the p-value of the respective sample with the associated control sample. *, P < 0.006; **, P < 0.0003. (C) Adhesion of Ovcar3 cells transfected with control (scrambled), RanBP2, or Epac1 siRNAs. 60 h after siRNA transfection, cells were allowed to adhere to a fibronectin-coated surface for 45 min in the absence or presence of 5 µM isoproterenol, and adhesion was subsequently detected by the measure of endogenous phosphatase activity. Shown are mean data with standard deviation from three individual experiments, which were normalized to the adhesion of control siRNA-transfected cells. Statistical analysis was performed using a Student’s t test. Asterisks indicate the p-value of the respective sample with the associated control sample. *, P < 0.02; **, P < 0.0003. The minus signs indicate unstimulated cells. IB, immunoblot.

Article Snippet: The following antibodies were used: mouse monoclonal GFP (Roche), Flag M2 (Sigma-Aldrich), rabbit polyclonal RanBP2 (Abcam), Rap1 (Santa Cruz Biotechnology, Inc.), and Ran (BD).

Techniques: Binding Assay, Activity Assay, In Vitro, In Vivo, Transfection, Control, Knockdown, Standard Deviation, One-tailed Test, Western Blot

Journal: PLoS ONE

Article Title: Molecular Characterization and Functional Analysis of Annulate Lamellae Pore Complexes in Nuclear Transport in Mammalian Cells

doi: 10.1371/journal.pone.0144508

Figure Lengend Snippet: (A) HeLa cells were treated with vinblastine or DMSO as a control and analyzed by immunofluorescence microscopy with mAb414 and anti-RanGAP1 antibodies. (B-F) HeLa cells were transfected with control or ELYS-specific siRNAs, double-labeled with mAb414 and anti-ELYS antibodies (B), mAb414 and anti-RanGAP1 antibodies (C), anti-importin α and anti-RanGAP1 antibodies (D), anti-importin β and anti-RanGAP1 antibodies (E), and anti-CRM1 and anti-RanGAP1 antibodies (F) followed by immunofluorescence microscopy. The boxes at the corner of each image represent the enlarged version of inlets. Bar, 10 μm.

Article Snippet: Antibodies used in this study were obtained from the following sources: anti-RanGAP1 (19C7) and anti-SUMO1 (21C7) mouse monoclonal antibodies (mAbs) [ ], Dr. Michael Matunis (Johns Hopkins, Baltimore, MD); anti-RanGAP1 rabbit polyclonal antibody (pAb) [ ], Dr. Mary Dasso (NIH, Bethesda, MD); anti-POM121 rabbit pAb (EMD Millipore); anti-tubulin mouse mAb (Sigma); anti-Myc (9E10) mAb (Santa Cruz); anti-lamin B goat pAb (Santa Cruz); anti-nucleophosmin rabbit pAb (Santa Cruz); anti-RanBP2 mouse mAb (Santa Cruz); anti-RanBP2 rabbit pAb (Abcam); mAb414 mouse mAb (Covance); anti-Myc rabbit pAb (Cell Signaling); anti-calreticulin rabbit pAb (Calbiochem); anti-ELYS mouse mAb (Bio Matrix Research); anti-ELYS rabbit pAb (Bethyl Laboratories); anti-importin α mouse mAb (BD Biosciences); anti-importin β mouse mAb (BD Biosciences); anti-CRM1 mouse mAbs (BD Biosciences; Santa Cruz); anti-FLAG mouse mAb (M2) (Sigma); anti-FLAG rabbit pAb (Sigma); anti-Ubc9 rabbit pAb (Abcam); anti-Ran mouse mAb (BD Biosciences).

Techniques: Immunofluorescence, Microscopy, Transfection, Labeling

Journal: PLoS ONE

Article Title: Molecular Characterization and Functional Analysis of Annulate Lamellae Pore Complexes in Nuclear Transport in Mammalian Cells

doi: 10.1371/journal.pone.0144508

Figure Lengend Snippet: HeLa cells were transfected with siRNAs specific to ELYS to upregulate annulate lamellae and then with the construct encoding Rev-GR-GFP fusion proteins. The transfected cells were incubated with LMB for 2 h to inhibit CRM1-mediated export, treated with both dexamethasone (1 μM) and LMB to induce importin α/β-mediated import for the indicated times, and analyzed by immunofluorescence microscopy. At least 50 Rev-GR-GFP cells were analyzed for each time point of dexamethasone treatment to select o representative cell as shown in this figure. The arrows indicated the sites of ALPCs. Bar, 10 μm.

Article Snippet: Antibodies used in this study were obtained from the following sources: anti-RanGAP1 (19C7) and anti-SUMO1 (21C7) mouse monoclonal antibodies (mAbs) [ ], Dr. Michael Matunis (Johns Hopkins, Baltimore, MD); anti-RanGAP1 rabbit polyclonal antibody (pAb) [ ], Dr. Mary Dasso (NIH, Bethesda, MD); anti-POM121 rabbit pAb (EMD Millipore); anti-tubulin mouse mAb (Sigma); anti-Myc (9E10) mAb (Santa Cruz); anti-lamin B goat pAb (Santa Cruz); anti-nucleophosmin rabbit pAb (Santa Cruz); anti-RanBP2 mouse mAb (Santa Cruz); anti-RanBP2 rabbit pAb (Abcam); mAb414 mouse mAb (Covance); anti-Myc rabbit pAb (Cell Signaling); anti-calreticulin rabbit pAb (Calbiochem); anti-ELYS mouse mAb (Bio Matrix Research); anti-ELYS rabbit pAb (Bethyl Laboratories); anti-importin α mouse mAb (BD Biosciences); anti-importin β mouse mAb (BD Biosciences); anti-CRM1 mouse mAbs (BD Biosciences; Santa Cruz); anti-FLAG mouse mAb (M2) (Sigma); anti-FLAG rabbit pAb (Sigma); anti-Ubc9 rabbit pAb (Abcam); anti-Ran mouse mAb (BD Biosciences).

Techniques: Transfection, Construct, Incubation, Immunofluorescence, Microscopy

Journal: PLoS ONE

Article Title: Molecular Characterization and Functional Analysis of Annulate Lamellae Pore Complexes in Nuclear Transport in Mammalian Cells

doi: 10.1371/journal.pone.0144508

Figure Lengend Snippet: ALPCs may serve as the docking or assembling sites for importin α/β-mediated import complexes followed by their dissociation for nuclear import. On the other hand, the ALPC-associated RanBP2/RanGAP1*SUMO1/Ubc9 complexes may function in the disassembly of CRM1-mediated export complexes by mediating RanGTP hydrolysis.

Article Snippet: Antibodies used in this study were obtained from the following sources: anti-RanGAP1 (19C7) and anti-SUMO1 (21C7) mouse monoclonal antibodies (mAbs) [ ], Dr. Michael Matunis (Johns Hopkins, Baltimore, MD); anti-RanGAP1 rabbit polyclonal antibody (pAb) [ ], Dr. Mary Dasso (NIH, Bethesda, MD); anti-POM121 rabbit pAb (EMD Millipore); anti-tubulin mouse mAb (Sigma); anti-Myc (9E10) mAb (Santa Cruz); anti-lamin B goat pAb (Santa Cruz); anti-nucleophosmin rabbit pAb (Santa Cruz); anti-RanBP2 mouse mAb (Santa Cruz); anti-RanBP2 rabbit pAb (Abcam); mAb414 mouse mAb (Covance); anti-Myc rabbit pAb (Cell Signaling); anti-calreticulin rabbit pAb (Calbiochem); anti-ELYS mouse mAb (Bio Matrix Research); anti-ELYS rabbit pAb (Bethyl Laboratories); anti-importin α mouse mAb (BD Biosciences); anti-importin β mouse mAb (BD Biosciences); anti-CRM1 mouse mAbs (BD Biosciences; Santa Cruz); anti-FLAG mouse mAb (M2) (Sigma); anti-FLAG rabbit pAb (Sigma); anti-Ubc9 rabbit pAb (Abcam); anti-Ran mouse mAb (BD Biosciences).

Techniques: