Fig. 2. TL-CAT is localized to the cell surface in stably transfected 3T3 cells. (a) As a positive control for flow cytometric analysis of GalT I expression on the cell surface, nontransfected 3T3 cells were incubated with antibodies raised against recombinant GalT I catalytic domain (-GalT I) and FITC-conjugated secondary antibody, and subsequently analyzed by flow cytometry as described in Materials and Methods. Control assays were incubated with preimmune serum (PI) rather than anti-GalT I antiserum. (b) 3T3 cells were stably transfected with CAT fusion constructs under the control of the inducible MT-1 promoter. Stably transfected 3T3 cells, induced with Zn²⁺, were labeled with anti-CAT antibody and FITC-conjugated secondary antibody, and analyzed by flow cytometry. The peak positions represent fluorescence intensity on the cell surface. Mock-transfected, or cells transfected with CAT alone or with TS-CAT show minimal fluorescent signals in both the uninduced (–) and induced (+) samples. Two populations of cells transfected with TL-CAT are shown in the mixed sample after Zn²⁺ induction (M+), representing adherent and nonadherent populations. The adherent cells (A+) show little surface staining above control levels (–) with anti-CAT antibody when induced with Zn²⁺ and assayed separately. However, fluorescence intensity increases dramatically in the population of live, nonadherent TL-CAT-expressing cells (F+).