Sitagliptin and the Blood-Retina Barrier: Effects on Retinal Endothelial Cells Manifested Only after Prolonged Exposure

Figure 9

Prolonged treatment of iBREC with sitagliptin decreased expression of CD29 and changed the amount of plasma membrane-localized CD9. (a) Proteins were immunoprecipitated from whole cell extracts with antibodies specific for CD9 or CD29 and analyzed by Western blot with an antibody binding to CD9. Precipitates obtained with both antibodies (αCD9 and αCD29) contained the tetraspanin CD9 but not samples derived from precipitation with an isotype-matched control antibody, indicating that CD9 and CD29 are present in the same protein complex. (b) Confluent iBREC exposed to 10-1000 nM sitagliptin for two days were harvested for preparation of cell extracts followed by Western blot analyses. Expression of CD29 and CD9 was lower after treatment with sitagliptin, although the differences were statistically significant only for CD29. Signals were normalized as described in Materials and Methods. (c) Prominent CD9-specific staining was observed at the plasma membrane of control cells (yellow arrows). This staining was more diffuse and less intense (yellow arrowheads) after treatment of the cells with the DPP-4 inhibitor for two days. Scale bar: 10 μm.

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