Nuclear CD26 Associates with a POLR2A-Related Genome Sequence Following YS110 Treatment.

<p>(A) Schematic diagram of chromatin immunoprecipitation (ChIP) cloning. JMN cells pretreated with YS110 for 3 hours were fixed, sonicated, and immunoprecipitated with Dynabeads to collect YS110-CD26-DNA complexes. The DNA fragments were cloned and identified by sequencing. The identity of candidate sequences was confirmed using data from GeneBank. (B) Genomic location of the CAS162 sequence. The 129-bp CAS162 sequence is located 894 bp downstream from the POLR2A gene. (C) ChIP analysis of CAS162 in JMN cells treated with control human IgG₁ or YS110 for 3 hours. Similar results were obtained in three independent experiments. (D) Biotin-labeled CAS162 oligonucleotide was used for electrophoretic mobility shift assay (EMSA). Nuclear extract (NE) was also collected from JMN cells pretreated with YS110 for 3 hours. After 20 minutes at room temperature the extracts, with or without recombinant CD26, were subjected to immunoblot analysis with streptavidin. Non-biotinylated CAS162 was used as a competitor. Arrow indicates the CD26-CAS162 oligonucleotide complex. (E) Biotin-labeled CAS162 oligonucleotide was used for EMSA. NE was collected from JMN cells. After 20 minutes at room temperature the extracts, with or without antibodies, were subjected to immunoblot analysis with streptavidin. Arrow indicates the CD26-CAS162 oligonucleotide complex. YS, YS110; Ig, IgG₁. (F) MSTO (mock or CD26_wt) cells were co-transfected with empty vector (pGL3) or CAS162 and phRL-TK, and relative luciferase activity was measured using a luminometer. Data were normalized for luciferase activity in cells transfected with phRL-TK, and are presented as mean values (± SD) from three independent experiments. *P<0.025. (G) JMN cells co-transfected with pGL3 or CAS162 and phRL-TK vector were incubated with control IgG₁ or YS110, and relative luciferase activity was measured using a luminometer. Data were normalized for luciferase activity in JMN cells transfected with phRL-TK, and are presented as mean values (± SD) from three independent experiments. *P<0.025.