Anti rna pol 2

Manufactured by Abcam

Sourced in United Kingdom

Anti-RNA Pol II is a laboratory reagent used to detect and study the RNA polymerase II enzyme, which is responsible for the transcription of protein-coding genes in eukaryotic cells. This antibody can be used in various applications, such as immunoprecipitation, Western blotting, and immunohistochemistry, to identify and quantify the presence of RNA polymerase II in biological samples.

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Lab products found in correlation

Anti phospho rna pol 2 ser2, by Abcam (2 mentions) Anti β actin, by Abcam (2 mentions) Ez magna chip g kit, by Merck Group (1 mentions) Anti meox2, by Santa Cruz Biotechnology (1 mentions) Complete mini, by Roche (1 mentions) Anti h3k27ac, by Abcam (1 mentions) Anti h3k4me3, by Abcam (1 mentions) Anti h3k27me3, by Abcam (1 mentions) Anti h3k9me3, by Abcam (1 mentions) Anti cdk7, by Cell Signaling Technology (1 mentions) Anti poly adp ribose polymerase anti parp, by Cell Signaling Technology (1 mentions) Anti cleaved parp, by Cell Signaling Technology (1 mentions) Phosphatase inhibitor, by MedChemExpress (1 mentions) Proteinase inhibitor, by MedChemExpress (1 mentions) Anti α tubulin, by Cell Signaling Technology (1 mentions) Pierce bca protein assay kit, by Thermo Fisher Scientific (1 mentions) Anti cdk1, by Abcam (1 mentions) Ripa buffer, by Sangon (1 mentions) Chip assay kit, by Merck Group (1 mentions) Anti h3k4me3 ab8580, by Abcam (1 mentions)

Spelling variants of this product

RNA Pol II (12 citations) Mouse anti-RNA pol-II (2 citations) Mouse anti-human RNA Pol II antibody (2 citations) Anti-phospho RNA Pol II (Ser2) (2 citations)

10 protocols using anti rna pol 2

Chromatin Immunoprecipitation of Lung Tumors

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FF lung tumor (LT; 3 mm³) samples consisting of almost 70% tumor tissue were pulverized under liquid nitrogen, fixed with 1% formaldehyde for 10 min, and immediately neutralized with 0.125 M glycine for 5 min. The resulting cells were washed with 1X PBS and treated with lysis buffer (50 mM HEPES-KOH pH 7.5, 140 mM NaCl, 1 mM EDTA pH 8, 1% Triton X-100, 0.1% sodium deoxycholate, and 0.1% SDS) containing 10 ml with protease inhibitors (complete Mini, Roche, Indianapolis, IN, USA).
The chromatin from the FF-LT samples was sonicated for 10 pulses of 20 seconds w/u 60 watts. Then, 10 μg of chromatin was immunoprecipitated (ChIP) using a commercial EZ-Magna ChIP™ G kit (Millipore, Temecula, CA, USA) and 2.5 μg of anti-MEOX2 (Santa Cruz Biotechnology, Dallas, TX, USA) as well as 1 μg of activated anti-H3K27Ac, anti-H3K4me3, anti-H3K27me3, anti-H3K9me3 or anti-RNA Pol II antibodies (Abcam, Cambridge Science Park, Cambridge, U.K.). A 1-μg aliquot of anti-mouse IgG was used as a negative control for ChIP (Millipore, Temecula, CA, USA).

Armas-López L., Piña-Sánchez P., Arrieta O., de Alba E.G., Ortiz-Quintero B., Santillán-Doherty P., Christiani D.C., Zúñiga J, & Ávila-Moreno F. (2017). Epigenomic study identifies a novel mesenchyme homeobox2-GLI1 transcription axis involved in cancer drug resistance, overall survival and therapy prognosis in lung cancer patients. Oncotarget, 8(40), 67056-67081.

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Western Blot Analysis of Cell Signaling Pathways

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Protein was extracted using RIPA buffer (Sangon Biotech, Shanghai, People’s Republic of China) containing proteinase inhibitors (MedChem Express) and phosphatase inhibitors (MedChem Express). The protein concentration was measured using the Pierce BCA Protein Assay Kit (Thermo Scientific). The following antibodies were used for Western blot: anti-CDK7 (Cell Signaling Technology, Danvers, MA, USA), anti-α-tubulin (Cell Signaling Technology), anti-poly(ADP-ribose) polymerase (anti-PARP) (Cell Signaling Technology), anti-cleaved PARP (Cell Signaling Technology), anti-RNA Pol II (Abcam, Cambridge, UK), anti-phospho RNA Pol II (Ser2) (Abcam), anti-phospho RNA Pol II (Ser5) (Abcam), anti-phospho RNA Pol II (Ser7) (Millipore), anti-CDK1 (Abcam), and anti-CDK1 (phospho T161) (Abcam).

Zhong S., Zhang Y., Yin X, & Di W. (2019). CDK7 inhibitor suppresses tumor progression through blocking the cell cycle at the G2/M phase and inhibiting transcriptional activity in cervical cancer. OncoTargets and therapy, 12, 2137-2147.

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ChIP Assay for Transcription Regulation

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The ChIP Assay Kit (Millipore) was used for ChIP analysis. Chromatin DNA was extracted and broken into fragments of 200–400 bp in length by sonication. The chromatin fragments were then immunoprecipitated with the following antibodies: IgG and anti-RNA pol II (Abcam). Both the precipitated DNA fragments and the genomic DNA were used for RT-PCR. By using the same primer pairs, the intensity of the PCR products from the precipitated DNA fragments was normalized against the intensity of the PCR products of the genomic DNA. Primers specific for the E2F1 promoter region (−202 to +25 bp) are listed in Table S7.

Yin J., Fu W., Dai L., Jiang Z., Liao H., Chen W., Pan L, & Zhao J. (2017). ANKRD22 promotes progression of non-small cell lung cancer through transcriptional up-regulation of E2F1. Scientific Reports, 7, 4430.

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Histone Modifications Analysis by Western Blot

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Western blots for histone modifications were performed on acid extracted nuclear proteins. The following antibodies used for western blot and ChIP assays were obtained from Abcam: anti-H3K4me2 (ab7766); anti-H3K4me3 (ab8580); anti-H3K18ac (ab1191); anti-H3K27ac (ab4729); anti-H3K36me3 (ab9050); anti-H3K79me2 (ab3594); and anti-RNA Pol II (ab817). Anti-H3K27me3 (07-449) was obtained from Millipore.

Wong S.H., Goode D.L., Iwasaki M., Wei M.C., Kuo H.P., Zhu L., Schneidawind D., Duque-Afonso J., Weng Z, & Cleary M.L. (2015). The H3K4-methyl epigenome regulates leukemia stem cell oncogenic potential. Cancer cell, 28(2), 198-209.

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Western Blot Analyses of Epigenetic Regulators

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Standard western immunoblotting procedures were used with the following antibodies: anti-H3K36me1 (Abcam), anti-H3K36me2 (Abcam), anti-H3K36me3 (Abcam), anti-SETD2 (Santa Cruz Biotechnology), anti-hMSH6 (Santa Cruz Biotechnology), anti-histone 3.3, anti-histone 3, anti-SKP2 (Santa Cruz Biotechnology), anti-biotin (Santa Cruz Biotechnology), anti-Ub (Santa Cruz Biotechnology), anti-P300 (Abcam), anti-RNA Pol II (Abcam), anti-CyclinE (Santa Cruz Biotechnology), anti-CDK4 (Santa Cruz Biotechnology), anti-CyclinD1 (Abcam), anti-PCNA (Abcam), anti-ppRB (Abcam), anti-E2F1 (Abcam), anti-P18 (Abcam), anti-P21/WAF1/Cip1 (Santa Cruz Biotechnology), anti-PKM2, anti-c-Myc (Santa Cruz Biotechnology), anti-Chk1 (Abcam), anti-P62 (Abcam), anti-KDM4A (Abcam), and anti-β-actin (Abcam).
The primers were as follows: P62: P1, 5′-GCAGTATCCCAAGTTCAATT-3′; P2, 5′-TGGGAACAGGTGGTGGAGGA-3′; P62 promoter: P1, 5′-GATCATTCACACCTGTGGAC-3′; P2, 5′-GGACGAGTGGTCACCCTCTG-3′; pre-miR-675: P1, 5′-CCCAGGGTCTGGTGCGGAGA-3′; P2, 5′-CCCAGGGGCTGAGCGGTGAG-3; mature mi675: P1, 5′-TGGTGCGGAGAGGGCCACAGUG-3′; U6 primer: P1, 5′-GCTTCGGCAGCACATATACT-3′; P2, 5′-GGAACGCTTCACGAATTTGC-3′; and β-actin: P1, 5′-CTTCCTTCCTGGGCATGGAG-3′; P2, 5′-TGGAGGGGCCGGACTGGTCA-3′.

Lu Y., Song S., Jiang X., Meng Q., Wang C., Li X., Yang Y., Xin X., Zheng Q., Wang L., Pu H., Gui X., Li T, & Lu D. (2018). miR675 Accelerates Malignant Transformation of Mesenchymal Stem Cells by Blocking DNA Mismatch Repair. Molecular Therapy. Nucleic Acids, 14, 171-183.

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Chromatin Immunoprecipitation for XBP1 and RNA Pol II

Cited 1 time

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Protein-protein cross linking was performed with DSG (2 mM, 45 min at 22°C) followed by protein-DNA cross linking with formaldehyde (34 (link)). Equal amounts of sheared chromatin were immunoprecipitated (IPed) overnight at 4°C. IPs were collected with 40 μL protein-A magnetic beads (Dynal Inc.), washed, and eluted in 250 µL elution buffer for 15 min at 65°C. Gene enrichment was determined by Q-gPCR using SYBR Green Master mix (Bio-Rad) and region-specific PCR primers (Table 2) at 500 nM. The fold change of specific genomic DNA in each two-step chromatin IP (XChIP) relative to control cells was calculated using the ΔΔC_t method (35 (link)) and normalized to the absolute amounts of input DNA for each XChIP (35 (link)). The ChIP-grade antibodies used in this study are anti-XBP1 (Abcam, Cat. No. ab37152) and anti-RNA Pol II (Abcam, Cat. No. ab26721).

Qiao D., Skibba M., Xu X., Garofalo R.P., Zhao Y, & Brasier A.R. (2021). Paramyxovirus replication induces the hexosamine biosynthetic pathway and mesenchymal transition via the IRE1α-XBP1s arm of the unfolded protein response. American Journal of Physiology - Lung Cellular and Molecular Physiology, 321(3), L576-L594.

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ChIP and MNase Assays for Epigenetic Profiling

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ChIP and MNase assays were performed as described55 56 (link). Briefly, 100 mg of eAT were cross-linked with 1% formaldehyde for 15 min at 37 °C. For ChIP assay, sonicated chromatin was immuno-precipitated with the following antibodies: anti-p300 (#SC-585, Santa Cruz Biotechnology), anti-Ac-H4-K16 (#07-329, Millipore, Temecula, CA), anti-DNMT1 (#NB100-56519) and anti-DNMT3b (#NB300-516) from Novus Biologicals (Littleton, CO), anti-DNMT3a (#ab2850), anti-MBD2 (#ab38646), and anti-RNA Pol II (#ab5408) from Abcam and anti-rabbit IgG (#I8140) and anti-mouse IgG (#I8765) from Sigma-Aldrich. For MNase assay, nuclei were isolated from 100 mg of eAT, suspended in wash buffer (100 mmol/L Tris-HCl, 15 mmol/L NaCl, 60 mmol/L KCl, 1 mmol/L CaCl₂) and treated with 200 U of MNase for 20 min at 37 °C. Cross-link reversal was performed at 65 °C for at least 16 h followed by an RNase and subsequent proteinase K digestion. DNA was purified by phenol–chloroform. Samples were then run on 1% agarose gel and the resulting mononucleosomal DNA fragments (~150 bp) were gel purified. For both assays, relative protein binding and nucleosome occupancy to the Ankrd26 gene were evaluated on recovered DNA by qPCR. Samples were normalized to their respective input using the 2^−ΔCT method.

Raciti G.A., Spinelli R., Desiderio A., Longo M., Parrillo L., Nigro C., D’Esposito V., Mirra P., Fiory F., Pilone V., Forestieri P., Formisano P., Pastan I., Miele C, & Beguinot F. (2017). Specific CpG hyper-methylation leads to Ankrd26 gene down-regulation in white adipose tissue of a mouse model of diet-induced obesity. Scientific Reports, 7, 43526.

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Detection of Influenza A Virus Proteins

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IAV proteins were detected using rabbit polyclonal antibodies anti-PB1 (GTX125923, GeneTex), anti-PB2 (GTX125926, GeneTex), and anti-NP (GTX125989, GeneTex) diluted 1:1000 in TBSTM [tris-buffered saline (TBS)/0.1% Tween 20 (Sigma-Aldrich)/5% milk]. Cellular proteins were detected using the rabbit polyclonal antibodies anti–glyceraldehyde-3-phosphate dehydrogenase (GTX100118, GeneTex) diluted 1:4000 in TBSTM and anti-RNA Pol II (ab5131, Abcam) diluted 1:100 in TBSTM; the mouse monoclonal antibodies anti-MAVS E-3 (sc-166583, Santa Cruz Biotechnology) diluted 1:200 in TMSTM and MitoTracker [113-1] (ab92824, Abcam) diluted 1:1000 TBSTM; and the rat monoclonal antibody anti-tubulin (MCA77G, Bio-Rad) diluted 1:1000 in TBSTM. Mouse monoclonal antibody anti-FLAG M2 (F3165, Sigma-Aldrich) diluted at 1:2000 was used to detect MAVS-FLAG. Secondary antibodies IRDye 800 donkey anti-rabbit (926-32213, LI-COR), IRDye 800 goat anti-mouse (926-32210, LI-COR), IRDye 680 goat anti-mouse (926-68020, LI-COR), and IRDye 680 goat anti-rat (926-68076, LI-COR) were used to detect Western signals with a LI-COR Odyssey scanner.

French H., Pitré E., Oade M.S., Elshina E., Bisht K., King A., Bauer D.L, & te Velthuis A.J. (2022). Transient RNA structures cause aberrant influenza virus replication and innate immune activation. Science Advances, 8(36), eabp8655.

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Western Blot Analysis of Cell Signaling Proteins

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HPASMCs were lysed in RIPA buffer (Thermo Scientific, Rockford, IL) supplemented with a protease inhibitor cocktail (Roche, Mannheim, Germany). Cell lysates were centrifuged at 12,000 rpm for 15 min at 4 °C, and the supernatants were then used as protein samples. Equal amounts of protein (30 μg) from each cell type were separated on SDS–polyacrylamide gels and transferred to nitrocellulose membranes (Bio-Rad, Munich, Germany). After blocking with 5% skim milk in Tris-buffered saline supplemented with 0.1% Tween 20 for 1 h at room temperature, the membranes were incubated at 4 °C overnight with the following antibodies: anti-CDK9 (CST, 1:500), anti-RNA pol II (Abcam, 1:1000), anti-phospho-RNA pol II (Ser-2) (Abcam, 1:1000), anti-Mcl-1 (Abcam, 1:1000), anti-c-Myc (Abcam, 1:1000), anti-survivin (CST, 1:500), and anti-β-actin (Abcam, 1:3000). The protein levels were normalized to β-actin. The gel bands were visualized with Amersham ECL prime Western blotting detection reagent (GE Healthcare, Little Chalfont, UK), and the band density was quantified with ImageJ software (National Institutes of Health, Bethesda, MD).

Jia Q., Hu Z., Song N, & Mao W. (2022). Flavopiridol Mitigates the Progression of Monocrotaline-Induced Pulmonary Hypertension in Rats by Targeting Cyclin-Dependent Kinase 9. Cardiovascular Drugs and Therapy, 37(3), 449-460.

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Quantitative Immunoprecipitation of FUS and RNA Pol II

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Immunoprecipitations were performed from cross-linked whole cell and nuclear lysates or from SEC fractions that were pooled from 9 to 15 mL. Pooled fractions were dialyzed into 0.02% SDS in TBS (IP buffer). IPs from cell lysates were diluted 1/10 into IP buffer. Fifteen micrograms of anti-FUS 4H11 (Santa Cruz Biotechnology, catalog no. sc-47711) or 5 μg of anti-RNA Pol II CTD4H8 (EMD Millipore, catalog no. 05-623) was incubated overnight at 4 °C with gentle rotation. Protein A/G beads (Pierce, catalog no. 20421) or Protein G Dynabeads (Invitrogen, catalog no. 10003D) were washed in IP buffer, and 50 μL (packed bead volume) was added to each sample. Incubation was continued for 2 h at room temperature. The beads were washed once with IP buffer, twice with IP buffer containing 500 mM NaCl, and twice with IP buffer. Protein was eluted by incubating beads with 2 × 100 μL of buffer [3.6 M MgCl₂ and 20 mM MES (pH 6.5)] and combining both eluates. FUS and RNA Pol II were detected in eluates and supernatants by ELISAs using polyclonal anti-FUS (Bethyl Laboratories, catalog no. A300-294A) or polyclonal anti-RNA Pol II (Abcam, catalog no. ab26721). The amount of protein recovered was determined by dividing the signals for eluted protein over the total (eluted + supernatant). The fraction of protein bound was corrected for the amount of targeted protein immunoprecipitated.

Thompson V.F., Victor R.A., Morera A.A., Moinpour M., Liu M.N., Kisiel C.C., Pickrel K., Springhower C.E, & Schwartz J.C. (2018). Transcription-Dependent Formation of Nuclear Granules Containing FUS and RNA Pol II. Biochemistry, 57(51), 7021-7032.

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