Analysis of HOPX-β methylation and expression in pancreatic cancer cell lines-1. (A) Schematic diagram of the 3 spliced transcript variants and common transcript core in HOPX (middle panel) and of CpG islands (gray area) in the 5′-flanking region of HOPX gene (bottom panel). Vertical bars indicate the dinucleotides CpG. Arrows indicate the sequences for bisulfite sequencing analysis or Q-MSP, respectively. F1, F2 and F3 represent forward primers for HOPX-α (331 bp) and HOPX-γ (456 bp), HOPX-β (376 bp), and HOPX-core (254 bp) in RT-PCR or Q-RT-PCR; R, common reverse primer; P, probe for Q-MSP; TSS, transcription start site; ATG, translation start codon. (B) Expression level of HOPX in PC cell lines was examined by RT-PCR (left panel) and Q-RT-PCR (HOPX-β and core/β-actin x 100, (right panel). (C) Expression level of HOPX in PC cell lines was examined by WB (top panel) and IP/WB (bottom panel). Transfectants we performed had the V5 epitope and polyhistidine region in the C-terminal peptide, and so, added approximately 5 kDa to original protein.