Ab64276

Manufactured by Abcam

Sourced in United Kingdom

Ab64276 is a laboratory equipment product. It is a device used for sample analysis in scientific research.

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8 protocols using ab64276

Immunostaining of Nuclear Pore Proteins

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For all immunostainings, primary antibodies (Abcam) used were rabbit anti-Nup358 at 1:200 (ab64276); rabbit anti-Nup214 at 1:200 (ab70497); mouse anti-Nup62 at 1:300 (ab96134); rabbit anti-Nup153 at 1:300 (ab171074); rabbit anti-TPR at 1:100 (ab170940); rabbit anti-lamin A at 1:200 (ab26300). Secondary antibodies: the secondary antibodies used were anti-mouse Cy3 (ab97035), anti-rabbit Cy3 (ab6939; Abcam), Alexa Fluor 647 goat anti-mouse (A21235), and Alexa Fluor 594 goat anti-rabbit (A11072; Molecular Probes).

Ashkenazy-Titelman A., Atrash M.K., Boocholez A., Kinor N, & Shav-Tal Y. (2022). RNA export through the nuclear pore complex is directional. Nature Communications, 13, 5881.

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Quantifying MX2 Protein Localization

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HeLa cells were transduced with puromycinR constructs bearing Flag-tagged wild-type or mutant MX2 cDNAs. Forty-eight h after transduction, stably expressing cells were selected with 1 μg/mL puromycin for 72 h and seeded onto coverslips at ~50,000 cells per well in 24-well plates; 24 h later, cells were washed with 1× phosphate buffer saline and fixed in 4% paraformaldehyde (EM Sciences) for 15 min. Cell membrane permeabilization was achieved by incubation with 0.2% Triton X-100 for 15 min. Then, the coverslips were blocked with NGB buffer (50 mM NH4Cl, 2% goat serum, 2% bovine serum albumin) for 1 h. MX2-Flag proteins were detected using anti-Flag monoclonal M2 and secondary antibody conjugated to Alexa 594 (Invitrogen, A21203). Endogenous NUP358 was detected using rabbit anti-NUP358 antibody (Abcam, ab64276) and secondary antibody conjugated to Alexa 488 (Invitrogen, A21206). The nuclei were labeled using DAPI (4’,6-diamidino-2-phenylindole) staining (0.1 mg/mL for 5 min). Cells were visualized using a Nikon A1 point-scanning laser confocal microscope (Nikon Instruments). Quantification of the nuclear envelope accumulation of wild-type or mutant MX2 proteins was calculated as the percentage of total protein colocalizing with NUP358, using Manders’ coefficient.

Betancor G., Bangham M., Jeon J.K., Shah K., Lynham S., Jimenez-Guardeño J.M, & Malim M.H. (2022). MX2 Viral Substrate Breadth and Inhibitory Activity Are Regulated by Protein Phosphorylation. mBio, 13(4), e01714-22.

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Multicolor super-resolution imaging of nuclear proteins

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Purified proteins were labeled for dSTORM using N-hydroxysuccinimidyl esters of Alexa649, Cy5, or additionally with Alexa 405/488/532 for multicolor STORM, according to the manufacturers' protocols. Antibodies were purchased from Abcam (Cambridge, UK): Anti-Nup153 antibody [SA1] [ab96462], Anti-RanBP2 antibody [ab64276], Donkey polyclonal Secondary antibody to Rabbit IgG—H&L [ab6701], Donkey polyclonal Secondary antibody to Mouse IgG—H&L [ab6707].

Lowe A.R., Tang J.H., Yassif J., Graf M., Huang W.Y., Groves J.T., Weis K, & Liphardt J.T. (2015). Importin-β modulates the permeability of the nuclear pore complex in a Ran-dependent manner. eLife, 4, e04052.

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Immunoblotting Assay for TRIM5 and Capsid Detection

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Dunni cells stably expressing HA-tagged TRIM5 variants were lysed in 100 mM NaCl, 50 mM Tris (pH 7.5) and 1% Triton X-100 containing protease inhibitors (Sigma-Aldrich). Proteins were separated on a 10% acrylamide gel, transferred onto PVDF membrane and probed with a rat anti-HA antibody (3F10, Roche Applied Science). A peroxidase-conjugated goat anti-rat (SouthernBiotech) was used as secondary antibody. Loading was controlled by probing with a β-actin antibody. For CA detection, conditioned cell-free supernatants of transfected HEK-293T cells were pelleted through a 20% sucrose layer in TEN buffer (10 mM Tris.HCl pH7.5; 100 mM NaCl; 1 mM EDTA) at 25000 rpm for 2h30 at 4 °C in a SW 40 Ti rotor. Pelleted viruses were probed for CA content using sera from HIV-2 infected patients and anti-human-IgG-HRP IgG (Sigma-Aldrich) as primary and secondary antibodies, respectively.
Protein depletion by shRNA in Jurkat cells was monitored by immunoblotting using a rabbit polyclonal antibody against Nup358/RanBP2 (ab64276, Abcam) and mouse Nup153 antibodies (ab24700, Abcam) with a peroxidase-conjugated goat anti-rabbit and anti-mouse as secondary antibodies, respectively (Sigma-Aldrich). Lamin-B was used as a loading control.

Mamede J.I., Damond F., Bernardo A.D., Matheron S., Descamps D., Battini J.L., Sitbon M, & Courgnaud V. (2017). Cyclophilins and nucleoporins are required for infection mediated by capsids from circulating HIV-2 primary isolates. Scientific Reports, 7, 45214.

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Antibodies for RanGAP1, Myc, and CRM1 Detection

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Antibodies used in this study include: mouse anti-RanGAP1 (19C7) monoclonal antibody (mAb) [14 (link)] from Dr. Michael Matunis (Johns Hopkins University, Baltimore, MD); mouse anti-Myc (9E10) mAb (sc-40; Santa Cruz); rabbit anti-Myc polyclonal antibody (2272; Cell Signaling); rabbit anti-RanBP2 polyclonal antibody (ab64276; Abcam); mouse mAb414 (MMS-120R; Covance); mouse anti-CRM1 mAb (611832; BD Biosciences).

Cha K., Sen P., Raghunayakula S, & Zhang X.D. (2015). The Cellular Distribution of RanGAP1 Is Regulated by CRM1-Mediated Nuclear Export in Mammalian Cells. PLoS ONE, 10(10), e0141309.

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Immunofluorescence Analysis of HIV-1 Capsid

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HIV-1 capsid protein p24 was stained using either anti-p24 AG3.0 (mAb from Dr. Jonathan Allan) or 183-H12-5C (HIV-1 p24 hybridoma from Dr. Bruce Chesebro) and were obtained through the NIH AIDS Reagent Program, Division of AIDS, NIAID, NIH. Rabbit polyclonal antibodies against Nup358 (ab64276) and Nup153 (ab84872) were purchased from Abcam. Rabbit anti-KHC to detect kinesin-1 heavy chain (sc-28538) were from Santa Cruz Biotechnology. Rabbit polyclonal to CPSF6 (ab99347) was purchased from Abcam and rabbit polyclonal to cyclophilin A (PA1-025) was purchased from ThermoScientific. Secondary antibodies conjugated to fluorophore for immunofluorescence studies were purchased from Jackson Immunoresearch Laboratories. Cyclosporine A (CsA; Sigma Aldrich) was used at a final concentration of 2.5 μM. DAPI to stain nucleus was obtained from Sigma Aldrich.

Dharan A., Talley S., Tripathi A., Mamede J.I., Majetschak M., Hope T.J, & Campbell E.M. (2016). KIF5B and Nup358 Cooperatively Mediate the Nuclear Import of HIV-1 during Infection. PLoS Pathogens, 12(6), e1005700.

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Antibody Characterization for Cell Biology

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The following antibodies were used:
Mouse α-tubulin (Sigma T5169, Western blot 1:10,000), mouse α-β-actin (Sigma A2228-100UL, Western blot 1:10,000), mouse monoclonal α-FXR1+2 (clone 2B12 from IGBMC, Western blot 1:1,000), rabbit α-FXR1 (Sigma HPA018246, immunofluorescence microscopy 1:800), mouse α-FXR1 (Millipore 03–176, immunofluorescence microscopy 1:800), mouse α-Nup133 (Santa Cruz sc-37673, Western blot 1:1,000), mouse α-FG-Nups (Abcam mAb414, immunofluorescence microscopy ab24609, 1:500), mouse cyclin B1 (Santa Cruz sc-245, clone GSN1, immunofluorescence microscopy 1:300, Western blot 1:2000), rabbit α-cyclin A (Santa Cruz sc-751, Western blot 1:1,000), rabbit α-cyclin D1 (Santa Cruz sc-718, Western blot 1:1,000), mouse cyclin E (Santa Cruz sc-247, Western blot 1:1,000), rabbit α-RanBP2 (Abcam ab64276, immunofluorescence microscopy 1:500, Western blot 1:1,000), rabbit α-Nup98 (Cell Signaling 2598S, Western blot 1:1,000), guinea pig α-p62 (Interchim GP62-C, immunofluorescence microscopy 1:500), rabbit α-p62 (Genetex GTX100685, Western blot 1:1,000), mouse α-ubiquitin P4D1 (Cell Signaling 3936, Western blot 1:1,000), and rabbit α-p-Rb (Cell Signaling 8516, immunofluorescence microscopy 1:1,600).

Agote-Arán A., Lin J, & Sumara I. (2021). Fragile X–Related Protein 1 Regulates Nucleoporin Localization in a Cell Cycle–Dependent Manner. Frontiers in Cell and Developmental Biology, 9, 755847.

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Quantifying MX2 Protein Localization

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HeLa cells were transduced with puromycinR constructs bearing Flag-tagged wild type or mutant MX2 cDNAs. 48 h after transduction, stably expressing cells were selected with 1 μg/ml puromycin for 72 h and seeded onto coverslips at ~50,000 cells per well in 24-well plates. 24 h later, cells were washed with 1x phosphate buffer saline and fixed in 4% paraformaldehyde (EM Sciences) for 15 min. Cell membrane permeabilization was achieved by incubation with 0.2% Triton X-100 for 15 min. Then, the coverslips were blocked with NGB buffer (50 mM NH4Cl, 2% goat serum, 2% bovine serum albumin) for 1 h. MX2-Flag proteins were detected using anti-Flag monoclonal M2 and secondary antibody conjugated to Alexa 594 (Invitrogen, A21203). Endogenous NUP358 was detected using rabbit anti-NUP358 antibody (Abcam, ab64276) and secondary antibody conjugated to Alexa 488 (Invitrogen, A21206). The nuclei were labelled using DAPI (4',6-diamidino-2-phenylindole) staining (0.1 mg/ml for 5 min). Cells were visualized using a Nikon A1 point-scanning laser confocal microscope (Nikon Instruments). Quantification of the nuclear envelope accumulation of wild type or mutant MX2 proteins was calculated as the percentage of total protein colocalizing with NUP358, using Manders' coefficient.

Betancor G., Bangham M., Jeon J.K., Shah K., Lynham S., Jiménez-Guardeño J.M., & Malim M.H. (2022). MX2 viral substrate breadth and inhibitory activity are regulated by protein phosphorylation.

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