Anti aff4

Cells were harvested and lysed on ice for 30 min in lysis buffer (50 mM Tris–HCl, 150 mM NaCl, 1 mM EDTA, 1% Nonidet P-40), supplemented with protease inhibitor cocktail (Pierce Biotechnology). Aliquots of the lysates were subjected to dodecyl sulfate-polyacrylamide gel electrophoresis. The resolved proteins were then transferred onto nitrocellulose membranes (Bio-Rad). The membranes were subsequently incubated with primary antibodies followed by a horseradish peroxidase-conjugated secondary antibody (Boster, Wuhan, China). Protein bands were detected using an enhanced chemiluminescence western blotting detection kit (Thermo). The following antibodies for western blot were used in this study: anti-AFF4 (Abcam, 1:1000), anti-SOX2 (Abcam, 1:2000), anti-HA-tag (Sigma–Aldrich, 1:2000), anti-α-tubulin (Sigma–Aldrich, 1:5000).

Chromatin immunoprecipitation (ChIP) assay was performed using a Simple ChIP Assay Kit (Cell Signaling Technology, Danvers, MA) according to the manufacturer’s instruction. The precipitated DNA samples were purified and measured by Q-PCR. Results were shown as the percentage of input controls. The following antibodies were used: anti-AFF4 (Abcam, 1:500), anti-CDK9 (Abcam, 1:500) and anti-H3K4me3 (Millipore, 1:1000). The following primers were used: 5′- CGTCACATGGATGGTTGTCTAT-3′ (forward) and 5′- GGCTCAAACTTCTCTCCCTTTC-3′ (reverse) in the SOX2 promoter region; 5′- TGGTGCAAAAACATCTTGGA-3′ (forward) and 5′- TACCCAAGAACCAGGAGTGG-3′ at around 10 kb downstream of SOX2 transcription start sites.