Anti-CD26 mAbs Enhance Nuclear Localization of CD26.

<p>(A) Nuclear (Nuc), cytoplasmic (Cyto), and membrane (Mem) fractions of MSTO cells stably transfected with empty vector (mock) or CD26 (clone8 and clone12) were prepared (Qiagen kit), immunoprecipitated with 1F7, and subjected to immunoblot analysis. Nuclear (Nuc) and cytosolic (Cyto) fractions of tumors from two malignant mesothelioma patients were prepared (Thermo kit), and subjected to immunoblot analysis. Lamin A/C, Calpain 1/2, and PDC-E2 and Na⁺/K⁺ ATPase were used as nuclear, cytoplasmic and membrane markers, respectively. (B) Immunogold staining of CD26 (15 nm gold particles, arrows) in ultrathin sections of JMN cells. Cyto, cytoplasm; Nu, nucleus. Scale bar, 200 nm. (C) Immunoblot analysis of CD26 in nuclear and cytosolic fractions of JMN cells treated with YS110 for the indicated times. Na⁺/K⁺ ATPase and Lamin A/C were used as cytosolic and the nuclear markers, respectively. (D) Diagram of each CD26 deletion mutant (left picture). CD26 contains a cytoplasmic domain (amino acids 1–6), a transmembrane domain (TM) (amino acids 7–29), a glycosylated domain (GD) (amino acids 85–321), and a dipeptidyl peptidase IV domain (DPPIV) (amino acids 627–740). Human embryonic kidney (HEK) 293 cells transiently expressing each flag-tagged construct were subjected to subcellular fractionation and immunoblot analysis with antibodies to Flag and nucleostemin (as a nuclear marker). (E) Immunofluorescence analysis of HeLa cells transfected with GFP-CD26_wt or GFP-CD26_1–629 and treated or not treated with Alexa-YS110 for 1 hour. In the merged image, GFP-fused proteins are shown in green, Alexa-YS110 is shown in red, and the nucleus is shown in blue. Arrows indicate colocalization of Alexa-YS110 and CD26_wt in the nucleus. Scale bars, 10 µm.