Subcellular fractionation technique was performed as we’ve previously described101 (link). Two million (2.0 × 106) serum-containing and serum-deprived PC3 and DU145 cells were stimulated with 250 μM H2O2 or TNFα (0.1 ng/mL) for 30 min. Alternatively, select samples were pre-treated with 5 mM NAC for 1 h prior to 250 μM H2O2 for 1 h. Cells were harvested for subcellular fractionation according to the manufacture’s protocol (NE-PER Nuclear and Cytoplasmic Extraction Kit, ThermoFisher). Briefly, cells were lysed in a series of buffers, centrifuged to obtain a non-nuclear fraction and an intact nuclear pellet, and further lysed to isolate the nuclear fraction. Forty micrograms (40 μg) of total cell lysate were resolved by SDS-PAGE to detect RelA/p65 (1:1,000, Cell Signaling Technology). Topoisomerase I (1:1,000, Santa Cruz Biotechnology) and β-actin (1:1,000, Cell Signaling Technology) were used as loading controls.
Topoisomerase 1
Manufactured by Santa Cruz Biotechnology
Sourced in United Kingdom, United States
Topoisomerase I is an enzyme that plays a crucial role in DNA replication and transcription by introducing transient single-strand breaks in DNA to relieve torsional stress. It functions by catalyzing the winding and unwinding of DNA, which is essential for various cellular processes.
Automatically generated - may contain errors
Lab products found in correlation
Rela p65, by Cell Signaling Technology (1 mentions)
Ne per nuclear and cytoplasmic extraction kit, by Thermo Fisher Scientific (1 mentions)
β actin, by Cell Signaling Technology (1 mentions)
Tubulin, by Merck Group (1 mentions)
Ecl substrate, by GE Healthcare (1 mentions)
Caspase 3, by Cell Signaling Technology (1 mentions)
Phospho enos s1177, by Cell Signaling Technology (1 mentions)
Myc tag, by Cell Signaling Technology (1 mentions)
Gapdh, by Abcam (1 mentions)
H3k27ac, by Diagenode (1 mentions)
Odyssey clx imaging system, by LI COR (1 mentions)
β actin, by Merck Group (1 mentions)
Fluorescent dye conjugated secondary antibodies, by LI COR (1 mentions)
A1978, by Merck Group (1 mentions)
β actin, by Santa Cruz Biotechnology (1 mentions)
4 protocols using topoisomerase 1
1
Subcellular Fractionation and Redox Response
2
Western Blot Analysis of Cellular Proteins
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After transfer of the proteins from polyacrylamide gels onto polyvinylidene difluoride membranes and blocking, membranes were incubated with antibodies directed against myc-tag (1:500), phospho-eNOS (S1177, 1:500) and Caspase-3 (1:3000 for full-length protein; 1:250 for the cleaved protein), all from Cell Signaling Technology, Frankfurt, Germany), GAPDH (1:70,000), eNOS (1:500) both from Abcam, Cambridge, UK, Topoisomerase I (1:200, Santa Cruz Biotechnology, Heidelberg, Germany), Tubulin (1:10,000; Sigma-Aldrich, Deisenhofen, Germany). Antibodies were incubated overnight at 4 °C. On the following day, membranes were incubated with secondary antibodies coupled to horseradish peroxidase, and detection was performed using ECL substrate (GE Healthcare, Solingen, Germany) and X-ray films.
3
Chromatin Modification Analysis by Western Blot
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Cells were lysed in Triton X buffer containing Tris-HCl (100 mM), NaCl (750 mM), NaPPi (50 mM), NaF (100 mM) Triton X-100 (10%), phenylmethylsulfonyl fluoride (PMSF, 1 mM), orthovanadate (OV, 2 mM), okadaic acid (OA, 10 nM) and protease inhibitor mix (PIM). The lysate was centrifuged (17,000 g, 10 min, 4 °C) and the supernatant was collected. Protein concentration was determined using Bradford assay. Samples were boiled in LämmLi buffer under reducing conditions, separated by SDS-PAGE and transferred to a nitrocellulose membrane by Western blotting. Fluorescence-based detection of secondary antibodies was performed using the Odyssey CLx imaging system (Licor, Bad Homburg, Germany). Primary antibodies: eNOS (#334600, Invitrogen), H3K27ac (#pAb-174-050, Diagenode), H3K27me2 (#C15410046-50, Diagenode) H3K27m3, H1 (#05–457, Millipore), H3 (#C15200011, Diagenode), mJmjD3 (#AP1022b, Abgent), hJmjD3 (#AP1022a, Abgent), UTX (#33510, Cell Signaling), EZH2 (#3147, Cell Signaling), topoisomerase I (#sc-5342, Santa Cruz) and β-actin (#A1978, Sigma-Aldrich). Fluorescence-labelled secondary antibodies: IRDye® RD680 Donkey anti-Mouse, RD680 Donkey anti-Rabbit, CW800 Donkey anti-Mouse, CW800 Donkey anti-Rabbit, RD680 Donkey anti-Goat (#926–68072, #926–68073, #926–32212, #926–32213, #926–68074 Licor).
4
Fractionation and Immunoblotting of Cellular Compartments
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For separation of nucleus and cytosol, the cells were lysed in buffer A (10 nM HEPES pH 7.9, 10 nM KCL, 0.1 mM EDTA, 0.1 mM EGTA, 1% Nonidet, 10 mM DTT, protein-inhibitor mix (PIM), 40 µg/mL phenylmethylsulfonylfluorid (PMSF). Cells were centrifuged to gain the cytosol containing supernatant. The pellet was cooked in sample buffer to gain the nuclear fraction. Bradford assay was used to determine the protein amount in the cytosolic fraction [19 (link)]. Samples were cooked in sample buffer and were transferred on SDS-PAGE followed by Western Blotting. Identical sample volumes from nuclear and cytosolic fractions were loaded. Analysis was performed with an infrared-based detection system using fluorescent-dye-conjugated secondary antibodies from LI-COR biosciences, Bad Homburg, Germany.
Primary antibodies used are: D7L7L from Cell signaling, Danvers, MA, USA for Cre-recombinase, A1978 from Sigma-Aldrich, St. Louis, MO, USA for β-actin and sc-5342 from Santa Cruz Biotechnology, Dallas, TX, USA for Topoisomerase I.
Primary antibodies used are: D7L7L from Cell signaling, Danvers, MA, USA for Cre-recombinase, A1978 from Sigma-Aldrich, St. Louis, MO, USA for β-actin and sc-5342 from Santa Cruz Biotechnology, Dallas, TX, USA for Topoisomerase I.
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