Anti smek1

For the ChIP assay, NPCs derived from wild-type or Smek1/2 dKO E11.5 forebrain or transfected with 4 μg pUltra-hot-Mbd3-flag or a pUltra-hot-empty vector for Mbd3 gain-of-function experiments and with pLKO3G-shMbd3 or pLKO3G-shScramble for Mbd3 loss-of-function were treated with 1% formaldehyde for 10 min at room temperature and quenched with 0.125 M glycine for ten more minutes at room temperature. Cross-linked chromatin was sonicated to fragment DNA to 200–1,000 base pairs, and then immunoprecipitation was performed with rabbit anti-IgG, anti-Smek1 (Sigma), anti-Mbd3 (Cell Signaling), anti-HDAC1, anti-HDAC2, anti-MTA1, and acetyl histone H3 (Santa Cruz Biotechnology) antibodies overnight at 4°C, followed by incubation with 50 μl of magnetic Protein A/G Dynabeads (EMD Millipore). Abundance of sequences in immunoprecipitates was determined by PCR and normalized as a fold-value relative to input chromatin. Smek ChIP-seq data were analyzed with the MACS online tool, and cis-regulatory sequences were analyzed using the Genomic Regions Enrichment of Annotations Tool (GREAT) interface (http://bejerano.stanford.edu/great/public/html/). We also utilized the Intergrative Genomics Viewer (IGV v2.3) to visualize distribution of ChIP-seq–identified peaks in different genomic regions. Primer sets for ChIP-qPCR are listed in S6 Table.

Cells were lysed by radioimmunoprecipitation assay lysis buffer (Bioteke) containing 1% protease inhibitor and 1% phosphatase inhibitors. The protein concentrations were determined by the bicinchoninic acid method (Thermo Scientific). The details of the immunoblotting assay were previously described. The following antibodies were used: anti-SMEK1 (Sigma, HPA002568), anti-phospho-STAT1 (Y701) (Cell Signaling Technology, #7649), anti-phospho-p65 (Cell Signaling Technology, #3039), anti-p65 (Cell Signaling Technology, #8242), anti-GAPDH (Proteintech, 60004-1-Ig), anti-STAT1 (Abways, AY4260), anti-AhR (Abways, CY3431), and anti-IDO1 (Santa Cruz, sc-365086). The lanes were analyzed by ImageJ.