Cloning and core region identification of SHP-1 P2 promoter. (A) Wild-type, full-length (− 802 bp ∼ + 157 bp, 960 base pairs) hematopoietic cell–specific SHP-1 P2 promoter was PCR amplified using primer pair F00 (forward primer, − 802 bp ∼ − 783 bp) and R00 (reverse primer, + 157 bp ∼ + 138 bp) at 1 × 94°C for 3 minutes, 30 × (94°C 30 seconds, 60°C 40 seconds, and 72°C 60 seconds), followed by 1 × 72°C 5 minutes. The amplified fragment was cloned into the pGL3-Enhancer vector, sequence confirmed, and named pwt-P2-Luc. The forward primers (F10 to F80) were subsequently designed so that a series of circa 100-bp 5′-truncated promoter fragments were achieved using the same reverse primer (R00). Similarly in panel B, a series of 3′-truncations was achieved using the same forward primer (F00) and different reverse primers (R10 to R80). (C) Structure of the full-length wild-type promoter (− 802 bp ∼ + 157 relative to the CAP site), large core (− 120 ∼ + 157), and small core (− 120 ∼ + 24), with putative transcription factor binding motifs labeled. (D) Promoter activity analysis of cloned SHP-1 promoter constructs through luciferase assays of the transient transfected Jurkat-LT cell lysates. (E) A comparison of the SHP-1 promoter activity in hematopoietic cell lines (Jurkat, Jurkat-LT, SupT1) and nonhematopoietic cell lines (HeLa, 293T). pGL3-Control and pCMV-Luc are plasmids that carry luciferase reporter gene driven by an SV40 promoter or a CMV promoter. Wt/LC/SC-P2-Luc: pGL3-based plasmid carrying luciferase reporter driven by the full-length wild-type, large core, or small core SHP-1 P2 promoter. Cell lysates were assayed in triplicates for luciferase activity and values represent the mean (± SD) of 2 experiments.