To generate enough material for Western blotting, cells were seeded at 175,000/15 cm plates (for samples collected up to 6 days after treatment) or at a density of 300 cells/well in 6-well plates (for samples at 9 days after treatment). Cell extracts were prepared in RIPA buffer and analyzed by 10% SDS-PAGE. Western blots were probed with anti-PARP (1:750; cat. no. 9542, Cell Signaling Technology); anti-γ-tubulin (1:1,000; cat. no. T-5192, Sigma-Aldrich); anti-eEF1A, eEF2 or RPL13a (rabbit, 1:1,000; Cell Signaling Technology); anti-cyclin D1 (rabbit, 1:250–500; cat. no. SC-753, Santa Cruz Biotechnology® Inc., Dallas, TX); anti-nucleolin (mouse, 1:5,000; cat. no. SC8031, Santa Cruz Biotechnology); anti-Cdk1 (1:1,000; cat. no. 9116, Cell Signaling Technology), anti-phospho-Y15 Cdk1 (1:1,000; cat. no. 9111, Cell Signaling Technology); anti-p21 (1:500; Cell Signaling Technology); anti-γ-H2Ax (1:250; Abcam); HRP-conjugated secondary antibodies and SuperSignal West Pico Solutions (Thermo Fisher Scientific Inc., Waltham, MA).
Anti nucleolin
Manufactured by Santa Cruz Biotechnology
Sourced in United States
Anti-nucleolin is a laboratory reagent that can be used to detect the presence of the nucleolin protein in biological samples. Nucleolin is a multifunctional protein involved in various cellular processes, including ribosome biogenesis, chromatin remodeling, and cell proliferation. The Anti-nucleolin product can be utilized in techniques such as Western blotting, immunoprecipitation, and immunohistochemistry to study the expression and localization of nucleolin in different cell types or tissues.
Automatically generated - may contain errors
Lab products found in correlation
Anti flag, by Merck Group (3 mentions)
Anti γh2ax, by Abcam (2 mentions)
Anti β actin, by Santa Cruz Biotechnology (2 mentions)
Anti ha, by Santa Cruz Biotechnology (2 mentions)
Anti caspase 3, by Cell Signaling Technology (2 mentions)
Anti cdk1, by Santa Cruz Biotechnology (1 mentions)
Anti cyclin d1, by Cell Signaling Technology (1 mentions)
Supersignal west pico solution, by Thermo Fisher Scientific (1 mentions)
Anti γ tubulin, by Cell Signaling Technology (1 mentions)
Rpl13a, by Cell Signaling Technology (1 mentions)
Anti p21, by Cell Signaling Technology (1 mentions)
Enhanced chemiluminescence detection reagent, by Elabscience (1 mentions)
Anti vinculin, by Santa Cruz Biotechnology (1 mentions)
Anti α tubulin, by Santa Cruz Biotechnology (1 mentions)
Anti b23, by Cell Signaling Technology (1 mentions)
Anti parp1, by Cell Signaling Technology (1 mentions)
Anti p21, by Santa Cruz Biotechnology (1 mentions)
Anti gapdh, by Cell Signaling Technology (1 mentions)
Dynabeads m 280 sheep anti mouse igg, by Thermo Fisher Scientific (1 mentions)
Anti akt, by Cell Signaling Technology (1 mentions)
9 protocols using anti nucleolin
1
Western Blot Analysis of Cellular Proteins
2
Immunoprecipitation and Western Blotting Analysis
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Immunoprecipitation assay was performed as previously reported48 (link). Briefly, whole cell lysate (1 mg) was incubated with 30 μl of protein A/G agarose beads coated with 5 μg of anti-uL3 (Primm, Milan, Italy) at 4 °C for 12 h. The beads were washed and boiled in the SDS sample buffer. The eluted proteins were loaded on 12% SDS-PAGE and detected by western blotting.
Western blotting analysis was performed as previously reported49 (link). The membranes were challenged with anti-E2F1 (Elabscience), anti-uL3 (Primm, Milan, Italy), anti-PARP-1, anti-GAPDH (Cell signaling), anti-B23, anti-p21, anti-α-tubulin, anti-β-actin, anti-nucleolin, anti-vinculin, anti-HA (Santa Cruz Biotechnology). Proteins were visualized with enhanced chemiluminescence detection reagent according to the manufacturer’s instructions (Elabscience®).
Western blotting analysis was performed as previously reported49 (link). The membranes were challenged with anti-E2F1 (Elabscience), anti-uL3 (Primm, Milan, Italy), anti-PARP-1, anti-GAPDH (Cell signaling), anti-B23, anti-p21, anti-α-tubulin, anti-β-actin, anti-nucleolin, anti-vinculin, anti-HA (Santa Cruz Biotechnology). Proteins were visualized with enhanced chemiluminescence detection reagent according to the manufacturer’s instructions (Elabscience®).
3
Coimmunoprecipitation of SLFN11 and RHA
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For coimmunoprecipitation (CoIP), H23 wild type cells were grown at 70% confluence. Nuclear extract was prepared with RIPA. Samples were pre-cleared using Dynabeads® M-280 Sheep Anti-Mouse IgG (Invitrogen), overnight at 4°C in rotation. Antibodies against SLFN11 (sc-374339, santacruz) and against RHA (sc-137198, santacruz) were pre-incubated with beads for two hours at 4°C in rotation. Samples were cleared of beads, and beads with the antibodies attached were recovered. Samples were incubated with their respective antibodies at 4°C in rotation for 2–4 h. After that, beads were washed three times with mild wash buffer (PBS plus 0.1% of NP40). Target proteins were then eluted using Laemmli buffer at 90°C for 10 min and elution proteins were separated from beads. To avoid cross-reactivity and unspecific band detection, mouse TrueBlot HRP secondary antibody was used. As a CoIP negative control we used normal mouse Ig from Millipore (12–371) or Anti-Nucleolin (sc-8031, Santacruz).
4
Molecular Signaling Pathway Analysis
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All chemicals were purchased from Sigma-Aldrich (Schnelldorf, Germany), if not indicated otherwise. Rapamycin came from Cell Signaling Technology (Frankfurt, Germany), LY294002 and SB203580 from Enzo Life Science (Lörrach, Germany). Antibodies were obtained from the following sources: anti-Akt and anti-phospho-S6 from Cell Signaling Technology (Frankfurt, Germany), anti-HA from Covance (Munich, Germany), anti-nucleolin from Santa Cruz Biotechnology (Heidelberg, Germany), and IRDyes 680LT and 800CW secondary antibodies from Li-COR Biosciences GmbH (Bad Homburg, Germany).
5
Characterization of HAUSP Protein Interactions
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A full-length cDNAs for human HAUSP, Mdm2, annexin-1, PKM2, PP2A, and p53 were PCR-amplified from HeLa and HCT116 and subcloned into the FLAG, Myc and GST epitope encoded vectors, respectively. The HAUSP (C223S) mutant was generated by site-directed mutagenesis. FLAG-tagged nucleolin constructs were kindly provided by Dr. Borowiec (New York University School of Medicine, USA). The HA-tagged ubiquitin, ubiquitin-K48 and ubiquitin-K63 constructs have been described47 (link). DNA constructs were transfected into cells by Lipofectamine 2000 (Invitrogen, Paisley, UK) according to the manufacturer’s instructions. Anti-Myc (1:200, 9E10 hybridoma cell media), anti-HA (1:200, 12CA5 hybridoma cell media), anti-FLAG (Sigma-Aldrich, St. Louis, MO, USA), anti-HAUSP, anti-p53, anti-nucleolin, anti-Mdm2, anti-β-actin, anti-ubiquitin (Santa Cruz Biotechnology, Santa Cruz, CA, USA), anti-γH2AX (Abcam, Cambridge, MA, USA) antibodies were used for immunoprecipitation, immunoblotting and immunofluorescence analysis.
6
Immunoblotting for Protein Analysis
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Immunoblotting was carried out using standard techniques. Briefly, cells were lysed in ice-cold 1x RIPA lysis buffer and protein concentrations determined. Aliquots (50μg) of protein were denatured in Laemmli loading buffer and separated on precast 4–10% NuPAGE Novex 4–12% Bis-Tris Protein Gels, (Novex-Invitrogen). The antibodies used for immunoblotting included anti—caspase3, p65, p50, Parp-1 RelB (Cell Signaling Technology, Danvers, MA), and Actin (Santa Cruz Biotechnology, Santa Cruz, CA). Nuclear extracts of the cells were prepared using the Nuclear Extraction Kit (Panomics, Redwood City, CA) and subjected to immunoblotting with anti-p65, -p50, (Cell Signaling Technology), and anti-nucleolin (Santa Cruz Biotechnology) antibodies.
7
Molecular Tools for SIRT7 Research
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Plasmids encoding hSIRT7, sh-hSIRT7, Flag-hSIRT7 (1 (link)), Flag/HA-SIRT7 and clonal lines that stably express Flag/HA-SIRT7 (13 ) have been described. SIRT7 truncation mutants were generated by PCR and cloned into pCMV-Flag vector. Oligonucleotides used for plasmid construction and mutagenesis are listed in Supplementary Table S1 . Expression vectors encoding HA-CDK9 and Flag-RPB1 were from Addgene. Antibodies against UBF, PAF53, RPA116 and SIRT7 have been described (13 ,17 (link),18 (link)). The following commercial antibodies were used: anti-acetyl lysine (Cell Signaling Technology, 9441), anti-actin (Abcam, ab8227), anti-CDK9 (Santa Cruz, sc-484 (C20)), anti-CHD4/Mi2 (Santa Cruz, sc-55606), anti-cyclin T1 (Santa Cruz, sc-10750), anti-DDX21 (Santa Cruz, sc-376953), anti-Flag (Sigma, F3165), anti-HEXIM1 (Bethyl Laboratories, A303-113A), anti-hnRNPK/J (Santa Cruz, sc-32307), anti-hnRNPU (Santa Cruz, sc-32315), anti-nucleolin (Santa Cruz, sc-13057), anti-nucleophosmin/B23 (Santa Cruz, sc-53175), anti-p53 (Abcam, ab31333), anti-Pol II (Santa Cruz, sc-56767 and sc-899 (N20)), anti-pSer2-Pol II (Active Motif, 6108), anti-pSer5-Pol II (Abcam, ab5408) and anti-tubulin (Sigma, clone B-5-1-2, T6074). anti-Flag M2 agarose was from Sigma (F1804). Secondary antibodies were from Dianova (111-035-144 and 115-035-062).
8
Western Blot Analysis of GATA4 Proteins
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Western blots of nuclear extracts from cardiac myocytes or other cell lines overexpressing various GATA4 proteins were performed as previously described.8 (link) Western blots of nuclear extracts from cardiac myocytes or other cell lines overexpressing various GATA4 proteins were performed as previously described.8 (link) Anti-HA (Santa Cruz, Santa Cruz, CA, USA, sc-805) anti-Flag (Sigma, F1804), anti-p300 (Santa Cruz, SC-585X) and anti-nucleolin (Santa Cruz, sc-55486) were all used at a dilution of 1/500. Anti-caspase-1 (Cell Signaling, Danvers, MA, USA, 2225), antiBclxL (Cell Signaling, 2762), anti-GAPDH (Abcam, Cambridge, UK, ab8245) and anti-GATA4 (Santa Cruz, sc-25310) were used at 1/1000 dilution. Homemade rabbit GATA4 and GATA6 antibodies were used at a dilution of 1/2000 and 1/500, respectively.38 (link)
9
Protein Expression Analysis by Western Blot
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Total protein content was extracted from cells using RIPA buffer (20 mMTris/HCl pH 7.2, 200 mMNaCl, 1% NP40) and Protease Inhibitor Cocktail and Phosphatase Inhibitor Cocktails II and III (Sigma-Aldrich). Samples were resolved in SDS-PAGE gels (NuPage 4-12% bis-tris Gel, Invitrogen). Protein expression was analyzed by standard western blot procedure using anti-Caspase 3 (Cell Signaling Technology, Danvers, MA, USA), anti-MCL1, anti-BCL-XL (both from Santa Cruz Biotechnology, Dallas, TX, USA), anti-PDK1, anti–PDK1_pS241, anti-PKCα/βII_pT638-41, anti Chk1 and anti-Chk1_pS345 (all from Cell Signaling Technology). The anti-nucleolin (Santa Cruz Biotechnology) or anti-β actin (Sigma Aldrich) antibodies were used as loading controls.
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