Fig. 3. Distinct upregulation of ADAM12 at the cell surface before onset of 3T3-L1 adipocyte differentiation. Growing cells are shown in (A) and (D), confluent (day 0) cells in (B) and (E), and maturing cells after 4 days of differentiation in (C) and (F). (A-C) Adherent cells were immunostained after permeabilization. (D F) Cells were immunostained in suspension without previous permeabilization, then subjected to cytospin centrifugation, as described in Materials and Methods. (G-I) ADAM12 mAbs were added to the cultures for 5 hours (G) 2 days (H) and 6 days (I), followed by detection of the ADAM12:IgG complex with a secondary Ab to visualize endocytosed ADAM12. The central red staining in the cells in panels A and B represent nonspecific nuclear immunoreactivity. Bars, 18 µm (A,B,D,E,G,H) and 30 µm (C,F,I).