Anti rna polymerase 2

The antibodies we used are as follows: anti-E-cadherin (Abcam # ab40772), anti-Vimentin (Abcam # ab92547), anti-Fibronectin (Proteintech # 15613–1-AP), anti-AGO2 (Abcam # ab57113), anti-USF1 (Santa Cruz # sc-390,027), anti-RNA polymerase II (Santa Cruz # sc-47,701), anti-TGF-β1 (Abcam # ab92486), anti-ESRP1 (Abcam # ab107278), anti-p-Smad2 (Cell Signaling Technology # 8828), anti-p-Smad3 (Cell Signaling Technology # 9520), anti-Smad2/3 (Cell Signaling Technology # 8685), anti-GAPDH (Proteintech # 10494–1-AP) and anti-β-actin (Cell Signaling Technology # 4970). Actinomycin D and RNase R were purchased from Sigma-Aldrich (St Louis, MO, USA) and Epicentre Technologies (Madison, WI, USA), respectively. The TGF-β receptor type I/II inhibitor LY2109761 was obtained from Selleckchem Chemicals (Houston, TX, USA).

All antibodies used in these experiments were either conjugated in-house or purchased as indicated. Anti-IL-4 (11B11), anti-IL-12 (C17.18), anti-IFNγ (AN17.18.24), and anti-CD4 (GK1.5) antibodies were purified from hybridoma supernatants at the German Rheumatism Research Center and used at 10 μg/ml final concentration. FITC-conjugated anti-IFNγ (AN18.17.24; BD Pharmingen, Heidelberg, Germany), phycoerythrin-conjugated anti-IL-4 (11B11; BD Pharmingen, and BVD4–1D11; Miltenyi Biotec, Bergisch Gladbach, Germany) were used for intracellular cytokine staining. For the chromatin immunoprecipitation, the following antibodies were used: anti-c-MAF, anti-RNA polymerase II, anti-p300 and anti-STAT6 (polyclonal rabbit IgG; Santa Cruz Biotechnology, Heidelberg, Germany), anti-NFATc2 and -NFATc1 (polyclonal rabbit IgG; ImmunoGlobe Antikörpertechnik GmbH, Himmelstadt, Germany), anti-GATA-3 (mouse monoclonal; Santa Cruz Biotechnology), anti-NF-kB (polyclonal goat IgG; Santa Cruz Biotechnology), anti-Brg1 (rabbit antiserum; Merck-Millipore, Darmstadt, Germany). For the image cytometry, anti-NFATc2 (rabbit monoclonal, clone D4B1, Cell Signaling Technology, Leiden, The Netherlands) and donkey anti-rabbit IgG (coupled to Alexa Fluor 647, Molecular probes A31573, Darmstadt, Germany) were used.

The following antibodies were used to perform ChIP: anti-H3K27Me3 (Cell Signaling, Cat. No: 9733), anti-BAF155 (Santa Cruz, Cat. No: sc-9746), anti-RNA polymerase II (Santa Cruz, Cat. No: sc-899), or anti-EZH2 (Cell Signaling, Cat. No: 5246). An isotype-matched IgG was used as a negative control^{40 (link),41 (link)}. ChIP DNA was analyzed by quantitative PCR against the promoter or a non-peak negative control region (2 Kb upstream of transcription starting site) of the indicated genes using the following primers: DAB2 peak Forward: 5′- GTGTTCGGGGAGAAGTCAAA-3′, DAB2 peak Reverse: 5′- ACGGATCTGTGAAACGAAGC-3′, DAB2 non-peak Forward: 5′-CGGGTTCACGCCATTCT-3′ and DAB2 non-peak Reverse: 5′-CACAGTGAAACCCTGTCTCTAC-3′; DLC1 peak Forward: 5′-AAAATTTCCAAGCGCCACTA-3′, DLC1 peak Reverse: 5′- ACACCGCCTTCTACCTTCCT-3′, DLC1 non-peak Forward: 5′-ACTCTGTCTTCGAGGAGGAAATA-3′ and DLC1 non-peak Reverse: 5′-ATCAGTGCCTAGGAGGAGTTAG-3′; NOXA peak Forward: 5′-TATTGTGGGAGGTGGGGATA-3′, NOXA peak Reverse: 5′- GGCCTGAAAACTTACGATGG-3′, DLC1 non-peak Forward: 5′- GCATTTCAGGGTGCGTATTTG-3′ and DLC1 non-peak Reverse: 5′- AAACCACTCCAGGCTCATTT-3′; PCR primers for the TIMP3 promoter are: TIMP3 Forward: 5′-ACTCCCCTACGCAAGGATTC-3′ and TIMP3 Reverse: 5′-CGTGTGAAGGCAGTTTGGTT-3′.