Fig. 2. BAD-LAMP is heavily glycosylated and is expressed after birth. (A) Immunoblot performed with a polyclonal antibody raised against the predicted peptide of the BAD-LAMP cytoplasmic tail. Lysates of mouse adult cortex and BAD-LAMP-transfected HeLa cells produced several bands migrating at above 35 kDa, whereas no reactivity is observed in control untransfected cells. The lowest form in the transfected cells is probably due to ER accumulation. (B) HeLa cells transfected with FLAG-BAD-LAMP cDNA were lysed and immunoprecipitated with anti-FLAG antibody. Immunoprecipitated material was treated with endoglycosidase H (endo H) or N-glycosidase F (N-gly F) prior to immunoblotting with anti-BAD-LAMP. In untransfected cells (NC), just the anti-FLAG IgG band (arrow) is observed, whereas several isoforms of BAD-LAMP were detected in transfected cells (Wt). Endo H treatment shows that the major band is endo H sensitive (gH) thus probably accumulating in the ER. The higher molecular mass bands were all N-gly F sensitive (gF). N-gly F treatment also demonstrated that all isoforms of BAD-LAMP are glycosylated and that the native molecular mass of the molecule is around 32 kDa (g0). The anti-FLAG IgG bands are also N-gly F sensitive, arrows. (C) After fractionation and isolation of cortical membranes, BAD-LAMP was found to be present exclusively in the membrane pellet (MbP) and not in the supernatant (Sup). Control syntaxin 6 and syntaxin 13 were also found in the membrane pellet, whereas RAB3, as expected, had a shared distribution due to its shuttling nature. (D) BAD-LAMP expression after birth. Mouse cortex lysates of different ages were immunoblotted with BAD-LAMP polyclonal antibody. BAD-LAMP expression levels are increased from birth to adulthood. (E) In situ hybridization for Bad-lamp on coronal post-natal brain sections from P2 to P12. Hemisections are presented. Bad-lamp expression appears at P2 in the cingulate cortex (arrowhead) and extends ventrally during the first post-natal weeks as a superficial and a deep band in the cortex. Subcortically, Bad-lamp is expressed transiently in the caudate putamen (cp). Bar, 500 µm.