ranbp2  (Azenta)

Journal: The Journal of Biological Chemistry

Article Title: A SUMO-interacting motif in the guanine nucleotide exchange factor EPAC1 is required for subcellular targeting and function

doi: 10.1016/j.jbc.2025.110279

Figure Lengend Snippet: RanBP2/Nup358 is essential for NE localization of EPAC1. A , time-dependent depletion of endogenous RanBP2/Nup358 by auxin. The expression levels of RanBP2 probed by immunoblotting analysis using anti-RanBP2 antibody in AID::NUP358 HCT116 cells treated with 1 mM auxin at 37 °C for various times. B , confocal imaging of AID::Nup358 HCT116 cells ectopically expressing EPAC1-mNG and treated with vehicle or Auxin for 3 h. mAb414 staining marks the nuclear envelope. Bar = 20 μm. AID, auxin-inducible degron; EPAC1, exchange protein directly activated by cAMP; NE, nuclear envelope; Nup358, nucleoporin 358; mNG, mNeonGreen.

Article Snippet: Primary antibodies used in this study are anti-EPAC1 antibody 5D3 (Cell Signaling Technology, Catalog no. 4155, 1:3000 dilution), anti-RanBP2 antibody (Santa Cruz Biotechnology, Inc, Catalog no. sc-74518, 1:1000 dilution), anti-Rap1 antibody (Santa Cruz Biotechnology, Inc, Catalog no. sc-65, 1:1000 dilution), anti-Rap2 antibody (BD Biosciences, Catalog no. 610215, 1:2000 dilution), and anti-SUMO2/3 antibody (MBL Life Science, Catalog no. M114-3, 1:3000 dilution).

Techniques: Expressing, Western Blot, Imaging, Staining

Journal: The Journal of Biological Chemistry

Article Title: A SUMO-interacting motif in the guanine nucleotide exchange factor EPAC1 is required for subcellular targeting and function

doi: 10.1016/j.jbc.2025.110279

Figure Lengend Snippet: The zinc finger domain of RanBP2/Nup358 is responsible for the NE localization of EPAC1. A , domain structures of the transfected Nup358 variants. B , subcellular localization of N-terminally 3×HA-tagged Nup358 variants ( red ), including Nup358 full-length (FL), NTD-ZFD, NTD-RanBD-1, NTD-OE, and EPAC1-mNG in AID::Nup358 HCT116 cells visualized by immunofluorescence confocal imaging after auxin-induced deletion of endogenous RanBP2. Bar = 20 μm. Line-scan plots show the quantified fluorescence intensities of EPAC1-mNG ( green ) and HA-tagged Nup358 variants ( red ) along the yellow lines crossing the nucleus, analyzed using ImageJ software. C , protein gel showing purified GST-Epac1 pulled down with RanBP2 ZFD using glutathione beads. AID, auxin-inducible degron; EPAC1, exchange protein directly activated by cAMP; NE, nuclear envelope; Nup358, nucleoporin 358; ZFD, zinc finger domain; mNG, mNeonGreen.

Article Snippet: Primary antibodies used in this study are anti-EPAC1 antibody 5D3 (Cell Signaling Technology, Catalog no. 4155, 1:3000 dilution), anti-RanBP2 antibody (Santa Cruz Biotechnology, Inc, Catalog no. sc-74518, 1:1000 dilution), anti-Rap1 antibody (Santa Cruz Biotechnology, Inc, Catalog no. sc-65, 1:1000 dilution), anti-Rap2 antibody (BD Biosciences, Catalog no. 610215, 1:2000 dilution), and anti-SUMO2/3 antibody (MBL Life Science, Catalog no. M114-3, 1:3000 dilution).

Techniques: Transfection, Immunofluorescence, Imaging, Fluorescence, Software, Purification

Journal: The Journal of Biological Chemistry

Article Title: A SUMO-interacting motif in the guanine nucleotide exchange factor EPAC1 is required for subcellular targeting and function

doi: 10.1016/j.jbc.2025.110279

Figure Lengend Snippet: EPAC1 SIM element is critical for EPAC1 and RanBP2 cellular interaction. A , the domain structure of transfected EPAC1 variants. B , association of endogenous RanBP2 with ectopically expressed EPAC1 constructs tagged with a C-terminal GFP in HEK293 cells probed by immunoprecipitation using an anti-GFP antibody. C , association of ectopically expressed GFP-tagged EPAC1 constructs with endogenous RanBP2 in HEK293 cells probed by immunoprecipitation using an anti-RanBP2 antibody. D , association of endogenous RanBP2 with ectopically expressed EPAC1 SIM variants tagged with a C-terminal GFP in HEK293 cells probed by immunoprecipitation using anti-GFP antibody. EPAC1, exchange protein directly activated by cAMP; SIM, SUMO-interacting motif.

Article Snippet: Primary antibodies used in this study are anti-EPAC1 antibody 5D3 (Cell Signaling Technology, Catalog no. 4155, 1:3000 dilution), anti-RanBP2 antibody (Santa Cruz Biotechnology, Inc, Catalog no. sc-74518, 1:1000 dilution), anti-Rap1 antibody (Santa Cruz Biotechnology, Inc, Catalog no. sc-65, 1:1000 dilution), anti-Rap2 antibody (BD Biosciences, Catalog no. 610215, 1:2000 dilution), and anti-SUMO2/3 antibody (MBL Life Science, Catalog no. M114-3, 1:3000 dilution).

Techniques: Transfection, Construct, Immunoprecipitation

Journal: The Journal of Biological Chemistry

Article Title: A SUMO-interacting motif in the guanine nucleotide exchange factor EPAC1 is required for subcellular targeting and function

doi: 10.1016/j.jbc.2025.110279

Figure Lengend Snippet: RanBP2 ZFD inhibits EPAC1 activation and EPAC1 activation by cAMP reduces its cellular interaction with RanBP2. A , in vitro basal and cAMP induced EPAC1 and EPAC2 GEF activity monitored in the presence or absence of RanBP2 ZFD. B , dose-dependent activation of EPAC1 by cAMP in the absence or presence of RanBP2 ZFD. C , cAMP binding reduces EPAC1 and RanBP2 cellular interaction. Association of endogenous RanBP2 with EPAC1 in HEK293 cells stably expressing EPAC1-V5-APEX2 probed by immunoprecipitation using anti-V5 magnetic beads in response to 007-AM treatment. D , 007-AM (5 μM) treatment reduces EPAC1 nuclear envelope localization. Snapshots and line-scan plots of live cell fluorescence confocal imaging of EPAC1-mNG in U-2 OS cells under basal and 007-AM treatment conditions. Bar = 10 μm. EPAC1, exchange protein directly activated by cAMP; ZFD, zinc finger domain; GEF, guanine nucleotide exchange factor; mNG, mNeonGreen.

Article Snippet: Primary antibodies used in this study are anti-EPAC1 antibody 5D3 (Cell Signaling Technology, Catalog no. 4155, 1:3000 dilution), anti-RanBP2 antibody (Santa Cruz Biotechnology, Inc, Catalog no. sc-74518, 1:1000 dilution), anti-Rap1 antibody (Santa Cruz Biotechnology, Inc, Catalog no. sc-65, 1:1000 dilution), anti-Rap2 antibody (BD Biosciences, Catalog no. 610215, 1:2000 dilution), and anti-SUMO2/3 antibody (MBL Life Science, Catalog no. M114-3, 1:3000 dilution).

Techniques: Activation Assay, In Vitro, Activity Assay, Binding Assay, Stable Transfection, Expressing, Immunoprecipitation, Magnetic Beads, Fluorescence, Imaging

Journal: The Journal of Biological Chemistry

Article Title: A SUMO-interacting motif in the guanine nucleotide exchange factor EPAC1 is required for subcellular targeting and function

doi: 10.1016/j.jbc.2025.110279

Figure Lengend Snippet: Schematic model of intracellular EPAC1 signaling sensitivity gradient. Interaction of EPAC1 with lipids on the plasma membrane or RanBP2 associated with the nuclear pore complex prime or impedes cAMP binding to EPAC1 at the respective cellular locus. EPAC1, exchange protein directly activated by cAMP.

Article Snippet: Primary antibodies used in this study are anti-EPAC1 antibody 5D3 (Cell Signaling Technology, Catalog no. 4155, 1:3000 dilution), anti-RanBP2 antibody (Santa Cruz Biotechnology, Inc, Catalog no. sc-74518, 1:1000 dilution), anti-Rap1 antibody (Santa Cruz Biotechnology, Inc, Catalog no. sc-65, 1:1000 dilution), anti-Rap2 antibody (BD Biosciences, Catalog no. 610215, 1:2000 dilution), and anti-SUMO2/3 antibody (MBL Life Science, Catalog no. M114-3, 1:3000 dilution).

Techniques: Clinical Proteomics, Membrane, Binding Assay