ChIPseq Smad3-JMJD3
ChIPseq Smad3-JMJD3
Neural stem cells grown with a media composed by equal parts of DMEM F12 (without Phenol Red, Gibco) and Neural Basal Media (Gibco) containing Penicillin/Streptomycin and Glutamax (1%), N2 and B27 supplements (Gibco), non essential aminoacids (0.1mM), sodium pyruvat (1mM), Hepes (5 mM), Heparin (2 mg/l), bovine serum albumin (25 mg/l) and ?mercaptoethanol (0.01 mM). We add fresh recombinant human EGF (R&D systems) and FGF (Invitrogen) to 20 ng/ml and 10 ng/ml final concentrations respectively e> we added TGFbeta1 30 minutes to the growth mediaChIPs from NSCs were carried out using previously described procedures {Frank, 2001 #110} with modifications: 3x106 NSCs untreated or treated with TGF? (5 ng/ml, for the indicated times) were fixed with di (N-succinimidyl) glutarate (DSG, Sigma) 0.2 mM, 45 min at room temperature followed of FA 1% 20 min. Fixation was stopped by addition of 0.125 mM glycine. The sonication step was performed in a Bioruptor sonicator (12 min, 30 sec ON, 30 sec OFF) and 1 mg of protein was used for each immunoprecipitation. Antibody-protein complex was captured with pre-blocked protein A (Amersham) and DNA purification was carried out using Nucleospin Extract II (Macherey-Nagel) columns.  ChIP DNA was analyzed by qPCR with SYBR Green (Roche) in a LightCycler 480 PCR system (Roche)
Experimental Factor: COMPOUND
TGF beta
Experimental Factor: IMMUNOPRECIPITATE
Smad3