Confluence-dependent upregulation of CD26 expression in HCT-116 and HCT-15. A) Confluence-dependent changes in CD26 protein expression were analyzed by Western blots. Cells (1 × 106) were plated in a 6-cm dish (day 0) and grown to post-confluence, with cells being confluent on day 3. Each lane was loaded with 50 μg protein. B) Densitometric analysis of the results from Western blots is shown. C) CD26 mRNA level was measured by real-time quantitative PCR as described in Materials and Methods. D) Immunofluorescence staining of CD26 in HCT-116 and HCT-15 cells on day 7 was performed as described in Materials and Methods. While fluorescence staining was not detectable at sub-confluence (data not shown), membrane localization of CD26 in both cell lines was demonstrated at post-confluence. All data shown are representative of three independent experiments.