JAML is a transmembrane protein induced upon ATRA-treatment in NB4 cells. (A) COS-7 cells were mock-transfected (-) or transfected with the p513-JAML-HA vector (JAML). Cell lysates were separated by SDS-PAGE and subjected to immunoblotting with anti-JAML (i) or anti-HA (ii) antibodies. The arrowhead indicates the major specific band. Brackets indicate minor specific bands likely corresponding to glycosylated JAML protein. (iii) JAML protein was in vitro–translated in the absence (–) or presence (+) of canine microsomal membranes. Arrow indicates a glycosylated JAML protein. (B) JAML protein expression during ATRA-induced differentiation of NB4 cells. NB4 cells were treated with 5 × 10^-7 M ATRA for different times. Protein lysates corresponding to 10⁵ cells were analyzed by Western blot, using anti-JAML or anti–β-actin polyclonal antibodies. In panels A and B, asterisks indicate nonspecific bands. (C) Cellular localization of JAML. MDCK cells stably transfected with the pMT-JAML-HA or pMT-JAMLK54D-HA were either untreated (-Zn) or treated (+Zn) with 125 μM ZnSO₄ for 40 hours. Cells were treated with anti-HA monoclonal antibody and stained with Cy3-conjugated goat anti-mouse antibody (ii,iii,vi, vii). In panels i, iv, v, and viii, the same cells as in panels ii, iii, vi, and vii, respectively, were counterstained with Hoechst 33258 reagent.