Figure 6. Thrombin stimulation of human corneal stromal fibroblasts and myofibroblasts induces the generation of multiple distinct extracellular N- and C-terminal Cyr61 forms. A-F: The conditioned media from fibroblasts and myofibroblasts cultured in serum-free medium alone (-) or 1.0 U/ml thrombin (+) for 24 h (A, D) or 3 h (B, C, E, F) were collected, processed, concentrated, and subjected to western blot analysis for Cyr61 with antibodies specific to the N-terminal (A, D), central linker (B, E), and C-terminal (C, F) regions of Cyr61 with antibodies specific to the N-terminal (A, D), central linker (B, E), and C-terminal (C, F) regions of Cyr61. Equivalent amounts of total protein were loaded in A and D, while samples in B, C, E, F were normalized based on GAPDH. Arrowheads indicate non-specific bands detected on secondary antibody only blots. Representative blots from two independent experiments using two different donors are shown. G-I: Approximate location of cleavage products based on epitope location of the Cyr61 antibodies.