Anti e2f4 sc 866

c17.2 cells were grown in an 80% confluence plate. Chromatin immunoprecipitation (ChIP) assays were performed as previously described [39 (link)]. Briefly, cells were lysed and chromatin from crosslinked cells was sonicated. Chromatin was incubated with 5 μg of anti-p27 (C-19, Santa Cruz), anti-p21 (sc-397, Santa Cruz) or anti-E2F4 (sc-866, Santa Cruz) in RIPA buffer (50 mM Tris - HCl pH 7.5, 150 mM NaCl, 1% NP-40, 0.5% Sodium deoxycholate, 0.1% SDS, 1 mM EDTA, 1 mM DTT, 1 mM PMSF, 0.1 mM Na₃VO₄, 0.5 μg/μl aprotinin, 10 μg/μl leupeptin) adding 20 μl of Magna ChIP Protein A magnetic beads (Millipore). Samples were incubated in rotation overnight at 4°C. Samples incubated without antibodies were used as a control. Beads were washed with low-salt buffer, high-salt buffer, LiCl buffer and TE buffer. Subsequent elution and purification of the immunoprecipitated DNA-protein complexes was performed using the IPure kit (Diagenode) according to manufacturer’s protocol. Samples were analysed by qPCR. Primer sequences used for qPCR of SNCA promoter genomic regions are listed in Supplementary Table 1.

Whole cell lysates were prepared at the indicated times after stimulation. Membranes were probed using the following antibodies: anti-IκBα (#9242, Cell Signaling, MA), anti-RelA (#3034, Cell Signaling, MA), anti-phospho-RelA (Ser 536) (#3031, Cell Signaling, MA), anti-IκBα (#9242, Cell Signaling, MA), anti-E2F-1 (#KH-95, Millipore Biotechnology, USA), anti-E2F-4 (sc-866, Santa Cruz), α-Tubulin Antibody (#2144 Cell Signaling, MA), and anti-cyclophilin A (#07–313, Millipore Biotechnology, USA).