Primary liver cells fixed in 4 methanol-free % PFA for 5 minutes were blocked and stained in IF buffer (1x PBS, 10 mg/ml BSA, 0.02% SDS, 0.1% Triton X-100). Primary and secondary antibodies was incubated for 1 hour at RT or overnight at 4°C with IF buffer washes between and after incubations. Cells were labeled with Hoechst 33342 (Life Technologies) for 5 minutes before mounting using VECTASHIELD (Vector Laboratories). Antibodies used: mAb414 (Covance or BioLegend); anti-Nup210 (Bethyl Laboratories, Inc.); anti-Pom121 (ThermoFisher Scientific); anti-Nup88 (22, BD Transduction Laboratories); anti-Nup107 (a kind gift from Martin Hetzer9 (link), The Salk Institute, La Jolla CA, USA); anti-Nup98 (39A3, Cell Signaling Technology); anti-Nup93 (F2, Santa Cruz BioTechnology); anti-Lamin A (Sigma Aldrich); anti-Lamin B1 (Abcam); anti-Cav1 (Cell Signaling Technology), and anti-Cav2 (65, BD Biosciences). Images were taken using a Leica SP8 confocal microscope and analyzed using the Leica Application Suite X software v3.1.5.16308, ImageJ v2.0.0-rc-54/1.51h (NIH), and Adobe Photoshop CS5.1 v12.1 x64.
Anti nup98
Manufactured by Cell Signaling Technology
Sourced in United States
Anti-Nup98 is a primary antibody that recognizes the Nuclear Pore Complex Protein Nup98. Nup98 is a key component of the nuclear pore complex, which is responsible for regulating the trafficking of molecules between the nucleus and cytoplasm.
Automatically generated - may contain errors
Lab products found in correlation
Anti lamin b1, by Abcam (2 mentions)
Anti lamin a, by Merck Group (2 mentions)
Anti pom121, by Thermo Fisher Scientific (2 mentions)
Anti nup210, by Fortis Life Sciences (2 mentions)
Anti nup88, by BD (2 mentions)
Anti nup107, by Cell Signaling Technology (2 mentions)
Anti nup93 f2, by Santa Cruz Biotechnology (2 mentions)
Anti cav2, by BD (2 mentions)
Anti cav1, by Cell Signaling Technology (2 mentions)
Hoechst 33342, by Thermo Fisher Scientific (2 mentions)
Sp8 confocal microscope, by Leica (2 mentions)
Mab414, by Fortrea (2 mentions)
Vectashield, by Vector Laboratories (2 mentions)
Anti lamin a c, by Cell Signaling Technology (2 mentions)
Anti p65, by Cell Signaling Technology (1 mentions)
Anti phosphorylated iκbα, by Cell Signaling Technology (1 mentions)
Criterion tris hcl gel, by Bio-Rad (1 mentions)
Anti tubulin, by Merck Group (1 mentions)
Anti iκbα, by Cell Signaling Technology (1 mentions)
Ne pertm nuclear and cytoplasmic extraction reagent, by Thermo Fisher Scientific (1 mentions)
7 protocols using anti nup98
1
Immunofluorescence Staining of Primary Liver Cells
2
Immunofluorescence Staining of Primary Liver Cells
3
Immunoblot Analysis of NMO Signaling Pathway
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Cells were serum-starved overnight prior to stimulation with NMO IgG or control IgG then lysed in RIPA buffer containing protease/phosphatase inhibitors. Cell lysates (10–30 μg) were run on 4–15 % Criterion Tris–HCl gels (Biorad). After transfer, blots were probed using anti-IκB-α (Cell Signaling 9242), anti-phosphorylated IκB-α (Cell Signaling 2895), anti-p65 (Cell Signaling 8242), anti-NUP98 (Cell Signaling 2598), or anti-tubulin (Sigma T9026) antibodies.
4
Fractionation and Immunoprecipitation of HBZ
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Nuclear and cytoplasmic fractions were prepared from ATL-2 (40x106 cells), Jurkat-HBZ expressing cells (5x106 cells) and Jurkat cells (5x106 cells) using NE-PERTM Nuclear and Cytoplasmic Extraction Reagents (Thermo Fisher Scientific) according to the manufacturer’s instruction. Five percent of nuclear and cytoplasmic extracts were resolved by 9% sodium dodecyl sulfate-polyacrilamide gel electrophoresis (SDS-PAGE) and analyzed by Western blotting with anti-Nup98 (Cell Signaling Technology) and anti-β-tubulin (Sigma-Aldrich) mAbs to assess the purity of nuclear and cytoplasmic fraction, respectively. HBZ proteins were precipitated from both nuclear and cytoplasmic fraction using the anti-HBZ 4D4-F3 mAb. Briefly, after preclearing with protein A-Sepharose beads and protein G-Agarose beads, cell extracts were incubated with anti-HBZ 4D4-F3 mAb for 1h and immunoprecipitated with protein A-Sepharose beads and protein G-Agarose beads overnight at 4°C.
5
Antibodies for Nucleoporin Analysis
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Antibodies used in this study included rabbit anti-Nup133–555 conjugated,40 (link) anti-Nup160,40 (link) anti-Nup53, anti-Nup85,40 (link) anti-Nup62 (Sigma-Aldrich, SAB1410438), anti-Nup214 (Abcam, ab84357), Anti-Nup98 (Cell Signaling Technology, #2598), anti-Nup93,83 (link) anti-Pom121 (Gene-Tex, GTX102128), anti-ELYS (Bethyl Laboratories, A300–166A), and mouse anti-FG Nup mAb414 (Biolegend, #mms-120p) and anti-LacI (Merck Millipore, 05–503).
6
Immunofluorescence Staining of Lymphoblastoid Cells
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The derived lymphoblastoid cells of the patient were fixed in 4% PFA at RT for 10 min, washed with PBS and cells were adhered to poly-Llysine coated glass slides by cytospin. Cells were subsequently permeabilized with 0.3% Triton X-100 in PBS for 5 min. Cells were blocked with 5% normal goat serum (Cell Signaling) in PBS for 1 h and then incubated with anti-NUP98 (Cell Signaling #2598T) or anti-lamin A/C (Cell Signaling #4777T) antibodies overnight at 4 • C. Slides were washed and incubated with anti-rabbit or anti-mouse (Alexa Fluor 488 or Alexa Fluor 594 Conjugate, Cell Signaling, 1:250) for 1 h at room temperature. After washing, the cells were mounted with an antifade mounting medium with DAPI (Vector Laboratories).
7
Quantitative Protein Assays in Cell Signaling
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Alanine Transaminase (ALT) and Aspartate Transaminase (AST) color endpoint assay kits were purchased from Bio Scientific (Austin, TX). BM chemiluminescence blotting substrate (POD) and 10X blocking reagent were obtained from Roche Diagnostics (Indianapolis, IN). iScript cDNA synthesis kit and SYBR Green super mix were bought from Bio-Rad (Hercules, CA). Mouse ultra-sensitive mouse insulin ELISA kits were purchased from Crystal Chem (Downers Grove, IL). High sensitivity CHIP kit was purchased from Abcam, Anti-Flag M2 mouse antibody was from Sigma Aldrich, Anti-insulin receptor (IR), anti-phospho-tyrosine (PY99), and HRP conjugated goat anti-mouse and goat anti-rabbit antibodies were from Santa Cruz Biotechnology (Santa Cruz, CA). Rabbit anti-phospho-AKT (Thr308), anti-phospho-AKT (Ser473), anti-AKT, anti-phospho-PERK (Thr980), anti-PERK, anti-Nup98, anti-Lamin A/C and anti-atubulin antibodies were purchased from Cell Signaling Technology (Beverly, MA). Tunicamycin and Thapsigargin were purchased from Sigma Aldrich (St. Louis, MO). Rabbit anti-phospho-IRE1(S724) was purchased from Abcam (Cambridge, UK).
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