N-glycosylation of CD40 mutants. (A) Immunoblot showing treatment of Control (C), an heterozygous carrier (P2′m) and CD40 mutants with PNGaseF. (B) A time-course treatment with PNGaseF was done for C and one of the mutants (P5), showing the appearance of a mono-glycosylated intermediate (empty arrowhead), and a complete deglycosylated product (solid arrowhead). (C) Immunoblot showing treatment of C and CD40 mutants with EndoH. P5 (right end panels) was analyzed after separation of cell surface biotinylated proteins from intracellular proteins. Vertical lines have been inserted to indicate repositioned gel lanes.