Frozen tissue sections were fixed with 10% formalin and cultured cells were fixed with 4% paraformaldehyde. All of the primary antibodies—Lamin A/C (Santa Cruz, Santa Cruz, CA, USA), Sun1 (Novus Biologicals), Sun2 (Abcam), Nesprin 2 (Abcam), γ-H2AX (Cell Signaling), and RhoA (Santa Cruz)—were used at a 1:100 to 1:300 dilution. All slides were analyzed via fluorescence microscopy (Nikon Instruments Inc. Melville, NY) and photographed at ×4–40 magnification. F-actin was stained with Alexa Fluor 488 Phalloidin or Alexa Fluor 594 Phalloidin (Thermo Fisher). The cell nuclei were stained with DAPI. Fibrosis formation in muscle tissues was visualized by Masson trichrome staining with the Trichrome Stain (Masson) Kit (Sigma-Aldrich). Sections were incubated in Weigert’s iron hematoxylin working solution for 10 min, and rinsed under running water for 10 min. Slides were transferred to Biebrich scarlet-acid fuchsin solution for 15 min before incubation in aniline blue solution for another 5 min. Slides were then rinsed, dehydrated, and mounted as earlier. The ratio of the area of fibrotic collagen (blue) to the area of normal muscle (red) was quantified to measure fibrosis formation.
Nesprin 2
Manufactured by Abcam
Sourced in United States, China
Nesprin 2 is a protein that is a component of the nuclear envelope. It plays a role in connecting the nucleus to the cytoskeleton.
Automatically generated - may contain errors
Lab products found in correlation
Alexa fluor 488 phalloidin, by Thermo Fisher Scientific (1 mentions)
Trichrome stain masson kit, by Merck Group (1 mentions)
γh2ax, by Cell Signaling Technology (1 mentions)
Alexa fluor 594 phalloidin, by Thermo Fisher Scientific (1 mentions)
Lamin a c, by Santa Cruz Biotechnology (1 mentions)
P erk1 2, by Cell Signaling Technology (1 mentions)
Fluorescence microscope, by Zeiss (1 mentions)
Erk1 2, by Beyotime (1 mentions)
Flag tag (flag), by Beyotime (1 mentions)
Hrp conjugated secondary antibody, by Thermo Fisher Scientific (1 mentions)
Chemidoc touch imaging system, by Bio-Rad (1 mentions)
Polyvinylidene difluoride membrane, by Merck Group (1 mentions)
Non fat milk, by Merck Group (1 mentions)
Surebeads protein g magnetic beads, by Bio-Rad (1 mentions)
β actin, by Merck Group (1 mentions)
Super signal femto reagent, by Thermo Fisher Scientific (1 mentions)
Gapdh, by Santa Cruz Biotechnology (1 mentions)
3 protocols using nesprin 2
1
Immunofluorescence and Fibrosis Analysis
2
Immunofluorescence Analysis of Cytoskeletal Proteins
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Cell climbing sheets were prepared and fixed in 4% paraformaldehyde for 10 min at room temperature. Subsequently, permeabilization was performed with 0.5% Triton X-100 for 10 min at room temperature. Additionally, three washes were performed with PBS for 5 min each, and the solution blocked with 5% BSA for 2 h. Primary antibodies against Flag (Beyotime, Shanghai, China, 1:200), Nesprin2 (Abcam, Cambridge, UK, 1:200), ERK1/2 (Beyotime, 1:200), and pERK1/2 (Cell Signaling Technology, Danvers, MA, USA, 1:100) were incubated at 4 ℃ overnight. After washing with PBS three times, a fluorescent secondary antibody was used and incubated at room temperature for 1 h. The nuclei were stained with DAPI, blocked with glycerol, and finally observed under fluorescence microscope (Zeiss, Jena, Germany). Each process was repeated three times.
3
Immunoprecipitation and Western Blot Analysis
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Extracts of primary cells and tumor tissues were prepared in an extraction buffer composed of 50 mM Tris-HCl (pH 8.0), 60 mM KCl, 10 mM MgCl2, 5 mM ATP, 4 mM EDTA, 1 mM dithiothreitol, 1% Nonidet P-40, 0.5 mM phenylmethylsulfonyl fluoride, and protease inhibitors cocktail and RIPA buffer, respectively, at 4°C. Cell lysates were sedimented at 10,000 × g for 10 min. NMIIA and NMIIB were immunoprecipitated with antibody specific to NMHCIIA (Abcam) and NMHCIIB (Covance), respectively, using SureBeads Protein G Magnetic Beads (Bio-Rad, CA) as per the manufacturer’s protocol. Immunoglobulin G (IgG) was used as negative control for immunoprecipitation. Immunoprecipitates, cell or tumor tissue lysates were fractionated by SDS–PAGE on 8% or 10% polyacrylamide Tris-glycine gels, and transferred onto 0.45 µm polyvinylidene difluoride membranes (Millipore) as previously published (Saha et al., 2011 (link); Das et al., 2015 (link)). Membranes were blocked with 5% BSA or nonfat milk (Sigma-Aldrich) and incubated overnight at 4°C with primary antibodies specific to NMIIA, NMIIB, α-tubulin, β-actin (Sigma-Aldrich), Nesprin-2 (Abcam), or GAPDH (Santa Cruz). Membranes were then incubated with HRP-conjugated secondary antibody (Thermo Fisher) for 2 h. Chemiluminescence signal was visualized by Super Signal Femto Reagent (Thermo Fisher) and captured using a ChemiDoc Touch Imaging system (Bio-Rad).
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