Chromatin Immunoprecipitation Assays

Chromatin immunoprecipitation (ChIP) assays were performed using the EZ-ChIP Immunoprecipitation Kit (Millipore) as previously reported.²¹ Anti-NFAT2 (ab2796) and anti-HDAC1 (ab7028) antibodies were used for immunoprecipitation. PCR was performed using primer pairs for the NFAT2 binding site: forward 5′-ACAGTGCCTCTGGTGC-3′ and reverse 5′-CCAAACCCAACTCCC-3′. ChIP assays detecting the recruitment of HDAC1 to the chitinase 3-like protein 1 (YKL40) and CD44 promoters were performed as previously described by Bhat et al.²³ The promoter regions of YKL40 and CD44 were determined using the National Center for Biotechnology Information gene database (https://www.ncbi.nlm.nih.gov/gene/?term) and Promoter 2.0 Prediction Server (http://www.cbs.dtu.dk/services/Promoter/). The promoters of YKL40 and CD44 were identified between −4000 bp upstream of the transcription start site and +100 bp downstream of the transcription start site. Ten PCR primers (Supplementary Table 2) were designed to examine the recruitment of HDAC1 to the YKL40 and CD44 promoters.

Additional details about the materials and methods are available in the Supplementary Materials.