Control rabbit igg antibody

ChIP with rabbit anti-CHOP (Cell Signaling Technologies) and control IgG rabbit antibody (Cell Signaling Technologies) was performed in Xbp1 and control silenced MODE-K cells by using a SimpleChIP Plus Enzymatic ChIP kit (Cell Signaling Technologies). Immunoprecipitated DNA was subject to qPCR to determine enrichment of CHOP binding to respective promoters (–431 to –272 bp relative to the start codon of Ulbp1), and results were normalized to input chromatin DNA. Primers used for qPCR were as follows: forward, 5′-TGTAGATCACCCTACCCAGCCT-3′ and reverse, 5′-TAAGAAGGACTCGAAGTGCAGGA-3′.

Chromatin immunoprecipitation (ChIP) was performed using the ChIP-IT^® Express Enzymatic
Kit (Active Motif, #53009, Carlsbad, CA, USA).
HSSYII and SYO-1 cells were grown to confluence in 15 cm dishes. Chromatin was cross-linked using 1% formaldehyde for 10 min, followed by glycine solution for 5 min to stop the reaction. Cells were then scraped into ice-cold PBS containing PMSF and centrifuged before incubation in lysis buffer for 45 min. Cells were then physically homogenized on ice to obtain nuclear lysates, which were incubated with digestion buffer for 5 min at 37°C, followed by incubation with the provided Enzymatic Shearing Cocktail for an optimized timepoint. Fifty microliters of the sheared lysate was used to confirm shearing efficiency and quantify DNA concentration. Ten micrograms of chromatin from each cell line was immunoprecipitated with either 2 µg of SS18-SSX antibody or control rabbit IgG antibody (Cell Signaling Technology, #2729, Danvers, MA, USA). Chromatin was then subjected to reverse cross-linking and proteinase K treatment, and subsequently stored at −20°C before qPCR assays.