Anti c jun

Chromatin Immunoprecipitation (ChIP) assays were performed essentially as described before (Li et al., 2018d (link); Li Z. et al., 2019 (link); Shao et al., 2019 (link); Weng et al., 2019 (link); Yang et al., 2019 (link)). In brief, chromatin in control and treated cells were cross-linked with 1% formaldehyde. Cells were incubated in lysis buffer (150 mM NaCl, 25 mM Tris pH 7.5, 1% Triton X-100, 0.1% SDS, 0.5% deoxycholate) supplemented with protease inhibitor tablet and PMSF. DNA was fragmented into ∼200 bp pieces using a Branson 250 sonicator. Aliquots of lysates containing 200 μg of protein were used for each immunoprecipitation reaction with anti-BRG1 (Santa Cruz, sc-10768), anti-p300 (Santa Cruz, sc-585), anti-c-Jun (Santa Cruz, sc-1694), anti-Fos (Santa Cruz, sc-166940), anti-SMAD3 (Abcam, ab28379), anti-ASH2 (Bethyl Laboratories, A300-489A), anti-JMJD2B (Bethyl Laboratories, A301-478), anti-anti-acetyl H3 (Millipore, 06-599), anti-trimethyl H3K4 (Millipore, 07-449), anti-trimethyl H3K9 (Millipore, 07-441), or pre-immune IgG. For re-ChIP, immune complexes were eluted with the elution buffer (1% SDS, 100 mM NaCO3), diluted with the re-ChIP buffer (1% Triton X-100, 2 mM EDTA, 150 mM NaCl, 20 mM Tris pH 8.1), and subject to immunoprecipitation with a second antibody of interest.

After isolation, content determination and electrophoresis, proteins were elettroblotted [46 (link)] and incubated with a polyclonal rabbit anti-CRBP-1, anti-Creb1, anti-CD44, anti-c-Jun, anti-Nox4, anti-p53, anti-RXRα, anti-RARα (Santa Cruz Biotechnology), anti-RARβ, (Abcam), anti-phosphorylated v-akt murine Jesi AN, Italy) and then sequenced using PyroMark Q24 thymoma viral oncogene homolog (pAkt Ser⁴⁷³), anti-AKT (pan), anti-phosphorylated extracellular-signal-regulated kinases (pErk1/2), anti-phosphorylated epidermal growth factor receptor (anti-EGFR Thr669), anti-EGFR antibody (Cell Signaling Technology, Danvers, MA, USA), anti-keratin 5 (clone H-40, Santa Cruz Biotechnology), mouse anti-vimentin (clone J144, Abcam), anti-keratin 14 (LL001, Santa Cruz Biotechnology) and anti-total tubulin antibody (Sigma-Aldrich). Revelation and densitometric blot analysis were performed in three independent experiments and Akt and EGFR activity expressed as phospho/total protein ratio [47 (link)].