Figure 5. Thrombin stimulation of human corneal stromal fibroblasts and myofibroblasts does not induce alternative splicing of the Cyr61 transcripts to produce the 24 kDa form of Cyr61. A and B: Fibroblasts (A) and myofibroblasts (B) in a serum-free medium were treated with (+) or without (-) 1.0 U/ml thrombin for 60 min. Total transcripts were isolated from the cells and PCR amplified using exon-exon bridging primer sets across the entire transcript length of Cyr61 (PCR program optimized for the full-length transcript) and were electrophoresed in 1.5% agarose gels with ethidium bromide. Samples were loaded for detection of PCR products rather than normalized for GAPDH. Representative gels are shown; n = 3 independent experiments with different cell donors. C: Diagram of human Cyr61 gene and locations of each primer set listed in Table 1. Black box = 5′ untranslated region, Green box = IGFBP domain, Blue box = vWFC, Pink box = TSP-1, Orange box = Knot, Gray box = 3ʹ untranslated region. Expected size of PCR products: 1–5 = 1,185 bp, 1–4 = 902 bp, 1–3 = 360 bp, 2–5 = 972 bp, and 2–4 = 689 bp.