The same strategy as described above was utilized to generate the R64, N79, and N77 mutant, in which all three amino acids were replaced for alanines. Briefly, a fragment with the mutated nucleotides with a 5 0 EcoRI site and a 3 0 XhoI site was synthesized by Genscript Corporation and replaced in the wild-type PSG1cDNA cloned into pFuse-IgG1 e3-Fc, previously digested with the same enzymes. The plasmids encoding the mutated PSG1s were transfected into CHO-K1 cells with Lipofectamine LTX (Invitrogen) according to the manufacturer's recommendations. Five hours posttransfection, the medium was aspirated, and the cells were kept in OPTI-MEM I (Invitrogen) for 72 h. The recombinant PSG1 G93D95-SL mutant, designated PSG1GDD!SDL, and the PSG1 R64N70N77-AAA mutant, designated PSG1RNN!AAA, were purified from the media using a protein A column as described above for wild-type PSG1-Fc. All proteins were determined to be endotoxin-free by their inability to induce tumor necrosis factor (TNF) in macrophages and with the HEK-Blue lipoplysaccharide (LPS) detection system (InvivoGen). The endotoxin levels in the proteins employed during these studies were tested, because LPS has been shown to induce VEGFA in some cell types.