Ptrchis

Full-length human IL-1α open reading frame was amplified by reverse transcriptase-polymerase chain reaction (RT-PCR) using the total RNA of HeLa cells. The fragment was cloned to the bacterial expression vector pTrc/His (Invitrogen, Waltham, MA, USA) with T/A cloning by adding the A residue during incubation with Taq polymerase (Takara, Shiga, Japan) and designated the pTrc-pIL-1α vector (Fig. 2a). This vector was used as a template to generate ppIL-1α and mIL-1α, which were then cloned to the same vector and designated pTrc-ppIL-1α (Fig. 2b) and pTrc-mIL-1α (Fig. 2c), respectively. The constructs were transformed to Escherichia coli (E. coli.) strain BL21 (Agilent, Santa Clara, CA, USA) and used for protein synthesis. In transfection experiments, ppIL-1α was cloned to green fluorescence protein (GFP)-containing mammalian expression vector pEGFP-C3 (Takara Bio USA, Mountain View, CA, USA) by an infusion method (In-Fusion HD Cloning Kit, Takara). The EcoRI site was added to the primers (pEGFP-ppIL-1α vector; Fig. 2d). The pEGFP-C3 vector was used as a control vector (Fig. 2e). pIL-1α, ppIL-1α, and mIL-1α were amplified by PCR and cloned to the EcoRI site of the pcDNA3.1 (Takara) expression vector (Fig. 2f,g,h,respectively). The sequences of all inserts were confirmed, and all plasmids were transformed to E. coli strain DH5α.