Association of PHF1 proteins with endogenous Gabrb1 in neurons. ChIP assays were performed using a PHF1(a and b) specific antibody and precipitated genomic DNA was found to contain the core promoter region of Gabrb1 in primary rat neocortical neurons. Detection of the endogenous Gabrb1 promoter was accomplished by PCR as depicted in (A) using two primers (arrows) that flank the β1-INR. The size of the PCR fragment is indicated above. Initiator position is depicted with a box and arrow showing the direction of transcription. (B) Bottom panel shows the presence or absence of Gabrb1-specific PCR products in fragments of genomic DNA that have been precipitated after addition of PHF1 antibodies. ChIP substrates are as indicated (1) primary neocortical neurons and (2) primary hippocampal neurons cultured for 7 days from E18 rat brains. (C) Representative data showing the presence or absence of Gabrb1-specific PCR products from ChIP performed with PHF1 antibody. Primary neocortical neurons were treated with either GABA (500 μM), GABA and the specific GABAAR antagonist bicuculline (50 μM), bicuculline alone, or relevant vehicle for 48 h, as described in Russek et al . Presence of IgG in reaction is represented as “-“ and PHF1 antibody as “+”. (D) Quantitation of ChIP data displayed in (C) is represented as mean ± SEM and expressed as percent increase from control (% control). (*=significantly different from control, p < 0.05). All samples were analyzed as ratios of PHF1 antibody/IgG after normalization to input.
Saha et al. BMC Pharmacology and Toxicology 2013 14:37 doi:10.1186/2050-6511-14-37