Figure 2

Biochemical analysis of the CD38 and CD157 molecules expressed by human corneal cells. (A) RT-PCR profile of CD38 and CD157 transcripts in corneal cells. PCR products were amplified from cDNA obtained from epithelial and stromal cells using CD38- or CD157-specific primer sets. Negative controls lacking cDNA template were used to rule out contamination. (B-C) Western blot analysis of corneal stromal and epithelial proteins immunoprecipitated with mAbs to CD38 and CD157. Precipitates of epithelial and stromal cells from biopsied corneas with anti-CD38 (IB4, IgG_2a) or with anti-CD157 (SY/11B5, IgG₁) mAbs were analyzed by 4% to 12% SDS-PAGE under non-reducing conditions and blotted onto PVDF membranes. Proteins with M_r of 45-kDa were immunodetected in the corneal epithelia and control Raji (CD38⁺) cells when blots were probed with anti-CD38 (AT13/5, IgG₁) mAb and developed by ECL (B). Proteins with M_r of 42- to 45-kDa were immunodetected in the stroma and corneal epithelia when blots were probed with anti-CD157 (SY/11B5, IgG₁) mAb and developed by ECL (C). NIH-3T3/CD157⁺ transfectants were used as positive control. Isotype-matched irrelevant mAbs were used as negative controls. (D) Ectoenzyme activity molecules assessed in epithelial corneal cells. Cells were incubated with 0.2 mM NAD⁺ or NGD⁺ at 37°C and samples collected after 0, 15, 60, and 120 min incubations. NADase (upper panel) and GDP-ribosyl cyclase (lower panel) enzymatic activities were determined by HPLC. Raji (CD38⁺) cells and NIH-3T3/CD157⁺ transfectants were used as positive controls. K562 (CD38⁻) cells and NIH-3T3 mock transfectants were the negative controls.