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ChIP assays were performed using reagents commercially obtained from Upstate, essentially according to the manufacturer’s instructions. The antibodies used in these experiments were rabbit polyclonal Ab against MKL1 (ab49311; Abcam) and anti-rabbit normal IgG (sc-2345, Santa Cruz). The amounts of each specific DNA fragment in immunoprecipitates were determined by PCR or quantitative PCR reactions. The fragment of mouse p21 promoter, containing the CArG box, was amplified using the forward primer: 5′-CCCTCGTGCTTAGACCA-3′, and reverse primer: 5′-GCTGTTGCTGCTACCCA-3′.

ChIP was performed as previously described, except extra crosslinking with 1.5 mM EGS for 30 min before addition of 1% formaldehyde [36 (link)]. Chromatin was incubated with anti-ZEB1 (5 μg, Santa Cruz H102, sc-25388X) and normal rabbit IgG control (5 μg, Santa Cruz, sc-2345) antibodies overnight and complexes were precipitated by protein A/G Dynabeads^® (Invitrogen 10002D/10004D, 25 μl each per IP). Precipitates were eluted (0.1 M NaHCO₃, 1% SDS) and chromatin was decrosslinked by first incubating 1 h at 37°C with 250 μg/ml RNaseA and 500 μg/ml proteinase K, followed by overnight incubation at 65°C. After DNA purification the indicated regions of NOG, FST and CHRDL1 promoters were amplified by quantitative PCR. HPRT1 was used as negative control [13 (link), 36 (link)].

Nuclear extracts were prepared from either pooled embryonic brains (E13.5) or adult cerebellum using the Nuclear Complex Co-IP Kit (54001, Active Motif, Carlsbad, CA, USA) as described previously (Harten et al., 2015 (link); Isbel et al., 2015 (link)). For each sample, 500 µg of nuclear lysate was incubated with 2 µg of anti-Wiz antibody (NBP180586, Novus Biologicals, Littleton, USA) or anti-IgG antibody (sc-2345, Santa Cruz Biotechnology, Dallas, TX, USA) overnight at 4 degrees Celsius. Immunoprecipitations were carried out using bead-conjugated Protein G (Dynabeads 10003D, Invitrogen, Mount Waverley, VIC, Australia) as per the Nuclear Complex Co-IP Kit manufacturer’s instructions. Eluted proteins were analysed via mass spectrometry at the La Trobe University Mass Spectrometry Facility, and the data were analysed as described previously (Harten et al., 2015 (link)).