Analysis of PAI-2 expression. (A) RT-PCR analysis of PAI-2 expression in HEK-293 cells. Clones expressing only GFP, or GFP-tagged exogenous ZNF198 and FGFR1 (^* indicates treated with bFGF) do not show detectable levels of PAI-2. In contrast, all 5 independently derived clones expressing ZNF198/FGFR1 (FUS) show PAI-2 expression. Clones 4 and 5 show reduced levels (see text). When these same cell clones were analyzed by Western blotting (B) the absence of PAI-2 was again seen in the cells carrying GFP, GFP-tagged ZNF, and GFP-tagged FGFR1 (bFGF stimulated), but all 5 clones carrying the ZNF198/FGFR1 gene showed 2 size variants of PAI-2, which were 47 and 32 kDa. A positive control from lipopolysaccharide-activated human monocytes was used to confirm the expression of the 2 molecular forms of PAI-2 in ZNF198/FGFR1-expressing clones. This control also demonstrates that the PAI-2 levels in cells expressing the fusion gene are not superphysiologic. When these same samples were probed with an anti-GFP antibody for Western blot analysis, the presence of the 177-kDa ZNF198 protein is detected in cells transfected with GFP-tagged ZNF198 and the smaller 172-kDa ZNF198/FGFR1 protein is seen in all of the 5 clones transfected with GFP-tagged ZNF198/FGFR1. The low levels of ZNF198/FGFR1 protein in clones 4 and 5 parallel the low levels of PAI-2 expression. Samples expressing the GFP vector alone showed only the 27-kDa band (not shown in the figure). (C) BaF/3 clones expressing the ZNF198/FGFR1 gene, detected by Western blotting using anti-GFP, also show induction of the PAI-2 mRNA and protein compared with parental BaF/3 cells, which do not. (D) Addition of bFGF to HEK-293 cells expressing the GFP-tagged FGFR1 gene does not result in the induction of PAI-2, although tyrosine phosphorylation of both the ZNF198/FGFR1 protein (arrow) and FGFR1 in FGFR1-expressing HEK-293 cells (100 kDa) is clearly seen.