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The cells were transfected with Setdb1 siRNA for 48 h. Approximately 30 μg protein was separated by 8–12% SDS-PAGE and transferred to PVDF membranes (Millipore). The membranes were probed using the following primary antibodies: NOX4 (1:500; NB110-58849; Novus), beta-Actin (1:2000; CW0096; CWBIO), SETDB1 (1:1000; 11231-1-AP; Proteintech), E2F1 (1:500; sc-193; Santa Cruz Biotechnology), FOXO4 (1:500; sc-5221; Santa Cruz Biotechnology), Lamin B (1:500; sc-6217; Santa Cruz Biotechnology), JNK (1:500; sc-7345; Santa Cruz Biotechnology), p-JNK (1:400; WL01813; WanleiBio), γH2AX (1:000; 2577; Cell signaling technology), p38 (1:500; sc-7972; Santa Cruz Biotechnology), and p-P38 (1:500; sc-17852-R; Santa Cruz Biotechnology). All were used as the manufacturer’s recommendation. The secondary antibodies were horse radish peroxidase-linked anti-mouse, anti-rabbit, or anti-goat IgG for 2 h at room temperature. The membranes were visualized on a Bio-Rad Chemidoc XRS using a Western Bright ECL Kit (Bio-Rad, Berkeley, CA, United States).

ChIP assays were based on the UpState protocol (http://www.tc.umn.edu/~muell002/Laboratory/protocols/X-ChIP.htm) using formaldehyde to crosslink genomic DNA of wild-type MEF cells. The chromatin was sheared to an average length of 300–500 bp. Monoclonal antibodies for Rb (Santa Cruz sc-50), E2F1 (Santa Cruz sc-193), E2F4 (Santa Cruz sc-866), and polyclonal antiserum for Zeb1 (a kind gift from Dr. Douglas Darling) were used for immunoprecipitation. Equal amounts of anti-IgG or pre-immune serum were used as controls. The sequence of primers for Ets1 promoter and the expected size of the PCR products are show in Additional file 2: Table S2. ChIP PCR reactions were similar to those described below for real-time PCR, but with additional 1% BSA and 1% DMSO, and the PCR programs usually had a higher annealing temperature (e.g. 60 – 68°C) and longer extension time (e.g. 1 minute).