Anti rabbit immunoglobulin g horseradish peroxidase linked secondary antibody

To determine the degree to which siRNA knockdown of CHD8 transcript results in decreased CHD8 protein, total protein was isolated using the Illustra triplePrep kit (GE Healthcare; Waukesha, WI, USA) and protein concentration was determined using the DC protein assay (Bio-Rad; Hercules, CA, USA). Total protein (10 μg) was then separated on a 4–20% gradient criterion TGX gel (Bio-Rad) and transferred to a nitrocellulose membrane by capillary transfer at 80 V for 3 h using a Bio-Rad Criterion blotter system. Blots were incubated overnight at 4 °C with anti-CHD8 (catalog no. 7656, Cell Signaling Technology; Danvers, MA, USA) and anti-GAPDH (catalog no. 1228, Cell Signaling Technology) primary antibodies diluted 1:1000 in blocking buffer. Anti-rabbit immunoglobulin G, horseradish-peroxidase-linked secondary antibody (catalog no. 7074, Cell Signaling Technology) was diluted 1:2000 and incubated with the membrane for 1.5 h at room temperature. Signal was developed using SuperSignal West Dura Extended Duration Substrate (Thermo Fisher Scientific; Waltham, MA, USA) and captured on a BioSpectrum Imaging System (UVP; Upland, CA, USA). The total density ratio of CHD8 to GAPDH was determined using the VisionWorks LS version 6.8 software (UVP) and analyzed by Student's paired t-test for comparison (n=4).