Antibodies used in this work were anti-SUN1 (ab124770, Abcam, used at 1/500); anti-nucleolin (ab136649, Abcam, used at 1/200); anti-Chk1 (sc8408, SantaCruz Biotechnologies, used at 1/500); anti-P-Ser345-Chk1 (2348LS, Cell Signaling, used at 1/1000); anti-P-Thr68-Chk2 (2661S, Cell Signaling, used at 1/1000); anti-Chk2 (05-649, Sigma-Aldrich, used at 1/1000); anti-calnexin (610523, Becton-Dickinson, used at 1/1000 for Western Blot and at 1/100 for immunofluorescence); anti-γ(pSer139]H2AX (05-636, Sigma-Aldrich, used at 1/500); anti-53BP1 (NB100-304, Novus, used at 1/500).
Ab124770
Manufactured by Abcam
Sourced in United Kingdom, China
Ab124770 is a polyclonal antibody that recognizes Glutathione S-Transferase (GST). It is suitable for use in various immunodetection applications, including Western blotting and ELISA.
Automatically generated - may contain errors
Lab products found in correlation
Ab124916, by Abcam (4 mentions)
Ab136649, by Abcam (1 mentions)
Nb100 304, by Novus Biologicals (1 mentions)
Sc 8408, by Santa Cruz Biotechnology (1 mentions)
Ab103021, by Abcam (1 mentions)
Ab198981, by Abcam (1 mentions)
Ubiquitin, by Santa Cruz Biotechnology (1 mentions)
β trcp, by Cell Signaling Technology (1 mentions)
Interleukin 4 (il 4), by Thermo Fisher Scientific (1 mentions)
α tubulin, by Merck Group (1 mentions)
Gm csf, by Thermo Fisher Scientific (1 mentions)
Ic5868p, by R&D Systems (1 mentions)
Lamin a c, by Santa Cruz Biotechnology (1 mentions)
Flag tag (flag), by Merck Group (1 mentions)
Cd11b, by Thermo Fisher Scientific (1 mentions)
M csf, by Thermo Fisher Scientific (1 mentions)
F4 80, by Thermo Fisher Scientific (1 mentions)
β actin, by Merck Group (1 mentions)
Gfp ab290, by Abcam (1 mentions)
Nup155, by GeneTex (1 mentions)
9 protocols using ab124770
1
Antibodies for Cell Signaling Analysis
2
Antibody Reagents for Cellular Analysis
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IFN-γ (315-05), IL-4 (214-14), M-CSF (315-02) and GM-CSF (315-03) were purchased from Peprotech (Rocky Hill, NJ). LPS (from Escherichia coli, serotype 055:B5, L4005) was purchased from Sigma (St. Louis, MO). The primary antibodies and dilutions used were: Flag (Sigma, F3165, 1:2000), α-tubulin (Sigma, T6199, 1:2000) and β-actin (Sigma, A2228, 1:5000), β-TrCP(Cell Signaling Technology, 4394, 1:1000), Nesprin1 (Santa Cruz, sc-99065, 1:500), Nesprin2 (Santa Cruz, sc-365097, 1:500), LaminA/C (Santa Cruz, sc-7292, 1:1000), ubiquitin (Santa Cruz, sc-8017, 1:1000), CD11b (eBioscience, 17-0112, 1:100), CD86 (eBioscience, 11-0862, 1:100), CD45 (eBioscience, 48-0451, 1:100), F4/80 (eBioscience, 25-4801, 1:100), CD206 (R&D systems, FAB2535P, 1:100) and Arg1 (R&D systems, IC5868P, 1:100) were obtained from. human SUN1 (Abcam, ab103021, 1:1000), mouse SUN1 (Abcam, ab124770, 1:1000) and mouse SUN2 (Abcam, ab124916, 1:1000), and mouse SUN2 (Abcam, ab198981, 1:100, FCS). An antibody specific for human SUN2 (1:1000) was produced by Shanghai Immune Biotech (Shanghai, China).
3
Western Blot Analysis of Cell Signaling Proteins
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After washing by ice-cold PBS, A549 and 95D cells were harvested and lysed using radioimmunoprecipitation assay-buffer for 1 hour at 4°C. After centrifuging at 13,000 rpm for 15 minutes, supernatant were collected, mixed with 4× protein loading buffer and treated for 10 minutes at 95°C. Protein samples were then separated by sodium dodecyl sulfate polyacrylamide gel electrophoresisand transferred to the polyvinylidene difluoride membrane. The membrane was incubated with primary antibody against SUN1 (#ab124770, 1:4,000 dilution; Abcam, Cambridge, UK), Cyclin D1 (#MD-17-3, 1:1,000 dilution; Medical & Biological Laboratories, Nagoya, Japan), CDK6 (#19117-1-AP, 1:500 dilution; Proteintech, Chicago, IL, USA), CDK2 (#2546, 1:1,000 dilution; Cell Signaling Technology, Danvers, MA, USA), CDK4 (#2906, 1:500 dilution; Cell Signaling Technology) or, glyceraldehyde 3-phosphate dehydrogenase (#10494-1-AP, 1:50,000 dilution; Proteintech) overnight at 4°C, followed by incubation of anti-rabbit or anti-mouse horseradish peroxidase-linked secondary antibody (1:5,000 dilution; Santa Cruz Biotechnology Inc., Dallas, TX, USA) for 1 hour at room temperature. Enhanced chemiluminescence reaction was performed using kit from the manufacturer (Amersham, Marlborough, MA, USA). Experiments were repeated at least three times.
4
Antibody sources and properties
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Nup153 antibody (hZ) was generated by the Ullman lab; antibodies against Nup133 and Elys were a kind gift from Douglass Forbes (University of California, San Diego). Nup96 and Nup98 were provided by the Powers lab. Antibodies against the SENPs and RanBP2/Nup358 were made in the Dasso lab. Other antibodies were obtained from commercial sources as follows: GFP (Ab290; Abcam, Cambridge, MA), importin α (610485; BD Transduction, San Jose, CA), Importin β (610559; BD Transduction), Importin 7 (SC-365231; Santa Cruz Biotechnology, Dallas, TX), 414 (MMS-120P; Covance, Princeton, NJ), Nup62 (610497; BD Transduction, Irvine, CA), POM121 (GTX 102128; GeneTex), RanGAP-1 (33-0800; Zymed), Nup155 (GTX120945; GeneTex), and Sun1 (Ab124770; Abcam).
5
Western Blot Analysis of SUN Proteins
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6
Immunofluorescent Labeling of Nuclear Envelope Components
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Cells were plated on untreated coverslips for experimental manipulation and fixed in either −20°C methanol for 5–10 min or 2% paraformaldehyde for 15 min. Primary and secondary antibody incubations were performed in blocking buffer for 1 h at room temperature. Primary antibodies are as follows: rabbit α-aurora B pT232 (Rockland; 600-401-677); mouse α-lamin A/C (Cell Signaling; 4C11); rabbit α-lamin B1 (Abcam; ab16408); mouse α-lamin B2 (Abcam; ab8983); hamster α-Lap2α (gift from Joe Glavy, University of Texas at Tyler); mouse α-Lap2β (gift from Brian Burke, Skin Research Institute of Singapore); rabbit α-LBR (Abcam; ab32535); rabbit α-Nup153 (Bethyl; 301-788A); mouse α-Nup153 (SA1; gift from Brian Burke); rabbit α-POM121 (GeneTex; GTX102128); rabbit α-SUN1 (Abcam; ab124770); mouse α-SUN2 (gift from Brian Burke); rat α-RFP (Proteintech; 5f8); rabbit α-tubulin (Abcam; ab18251); rat α-tubulin (Abcam; ab6160); chicken α-tubulin (Synaptic Systems; 302 206); sheep α-tubulin (Cytoskeleton; ATN02); followed by AlexaFluor conjugated secondary antibodies (ThermoFisher). Coverslips were mounted on slides using DAPI Prolong Gold, per manufacturer’s instructions (Invitrogen).
7
Antibody Validation for SUN and Myosin Proteins
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Primary antibodies were anti-SUN1 antibody (produced in rabbit; Abcam; ab124770; 1:1,000), anti-SUN2 antibody (rabbit; Abcam; ab124916; 1:1,000), anti-MIIA antibody (rabbit; Sigma-Aldrich; M8064; 1:1,000), and anti-MIIB antibody (N-17; goat; Santa Cruz Biotechnology; SC-47205; 1:7,500). β-Actin was used as a loading control (Purified Mouse Anti-Actin Ab-5; BD Biosciences; 612656; 1:10,000).
8
Western Blot Analysis of SUN1 and SUN2
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Cells were lysed with radio-immunoprecipitation assay lysis buffer (APExBIO, K1020), supplemented with protease inhibitor cocktail (APExBIO, K1007) and phosphatase inhibitor cocktail (APExBIO, K1015). Insoluble cell debris was removed by 15-min centrifugation at 13,500×g at 4 °C. Protein concentration was determined using a bicinchoninic acid protein assay kit (Beyotime, P0010S). Western blot was performed on total protein extracts. Proteins were then transferred to 0.45-μm polyvinylidene difluoride membranes (Sigma, IPVH00010) after electrophoresis. Membranes were blocked for 1 h at room temperature in blocking buffer (Beyotime, P0023B) and incubated overnight at 4 °C with antibodies in primary antibody dilution buffer (Beyotime, P0023A). Goat-anti-Rabbit horseradish peroxidase-conjugated (Abcam, ab2057181:10,000) antibody was used as secondary antibody. Protein bands were visualized with Clarity Western ECL Substrate (Bio-Rad, USA) and the pictures were obtained by ChemiScope Western Blot Imaging System (Clinx Science Instruments, China).
Primary antibodies and corresponding concentrations were used as follows: rabbit monoclonal anti-SUN1 (Abcam, ab124770, 1:1000) and rabbit monoclonal anti-SUN2 (Abcam, ab124916, 1:1000). Glyceraldehyde 3-phosphate dehydrogenase (Abcam, ab8245, 1:1000) was used as a loading control.
Primary antibodies and corresponding concentrations were used as follows: rabbit monoclonal anti-SUN1 (Abcam, ab124770, 1:1000) and rabbit monoclonal anti-SUN2 (Abcam, ab124916, 1:1000). Glyceraldehyde 3-phosphate dehydrogenase (Abcam, ab8245, 1:1000) was used as a loading control.
9
Co-Immunoprecipitation of Nuclear Envelope Proteins
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Co‐immunoprecipitation was performed as described previously.27 , 28 , 30 , 31 Cell lysates were mixed with protein A/G‐magnetic beads (Novex, Oslo, Norway) and incubated at 4°C overnight with the selected antibodies. The beads were washed using Western/IP lysis buffer (Beyotime, Haimen, China, composition: 20 mM Tris [pH 7.5], 150 mM NaCl, 1% Triton X‐100, and inhibitors containing sodium pyrophosphate, β‐glycerophosphate, EDTA, Na3VO4 and leupeptin), and eluted using the Acid Elution buffer (Beyotime). Next, total protein amount of each group was measured by BCA protein quantification. After the first quantification, the samples were diluted according to the difference in protein amount, and the second quantification was carried out for confirmation. Finally, the samples were suspended in SDS‐PAGE loading buffer and then measured by IB. The antibodies used for co‐IP were as follows: anti‐FLAG (CST, #14793 and #8146), anti‐PDHA (Abcam, #ab168379 and #ab110330), anti‐HA (Abcam, #ab9110 and #ab1424), anti‐EMD (Abcam, #ab40688 and #ab204987), anti‐Lamin A (Abcam, #ab226198), anti‐Nesprin1 (Abcam, #ab192234), anti‐Nesprin3 (Abcam, #ab186746), anti‐Sun1 (Abcam, #ab124770) and anti‐Sun2 (Abcam, #ab124916).
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