Transcytosis is used alone (e. transcytotic transportation of exogenous polymeric immunoglobulin Mrc2 receptor and endogenous glycosylphosphatidylinositol-anchored proteins CD59 to the apical membrane. MAL2 depletion did not affect the internalization of these molecules but produced their accumulation in perinuclear endosome elements that were accessible to transferrin. Normal transcytosis persisted in cells that expressed exogenous MAL2 designed to resist the depletion treatment. MAL2 is therefore essential for transcytosis in HepG2 cells. gene expression in hepatoma HepG2 cells and in the epithelial MDCK Ambrisentan kinase activity assay and Caco-2 cell lines, all of which use the transcytotic pathway to a greater (HepG2) or lesser extent (MDCK and Caco-2) to target membrane proteins to the apical surface. Since we Ambrisentan kinase activity assay considered the hypothesis that MAL2 may act as machinery for transcytosis plausible, we undertook a study of MAL2 function in hepatoma HepG2 cells, which are a paradigm for the study of the transcytotic pathway in a cellular context deprived of other apical routes of transport for single transmembrane and glycosylphosphatidylinositol (GPI)*-anchored proteins (Bastaki et al., 2002). Results and discussion The MAL2 protein resides in lipid rafts in hepatoma HepG2 cells Human MAL2 is a 176-residue (gene in different cell lines. Total RNA from the indicated cell lines was hybridized to DNA probes specific to MAL2, MAL, or -actin. (C) Characterization of a novel mAb to MAL2. To assay the specificity of mAb 9D1, protein extracts from untransfected (U) or from transfected (T) COS-7 cells transiently expressing MAL2 tagged with the c-Myc 9E10 epitope were subjected to immunoblot analysis with either mAb 9D1 or the antitag mAb 9E10. Since COS-7 cells are negative for gene expression (unpublished data), no reaction was observed with endogenous proteins of COS-7 cells. (D) mAb 9D1 detects endogenous MAL2. Membrane fractions from the indicated cell lines were subjected to immunoblot analysis with anti-MAL2 mAb 9D1. (E) Identification of endogenous MAL2 in lipid raft fractions in HepG2 cells. Cells were extracted with 1% Triton X-100 at 4C and subjected to centrifugation to equilibrium in sucrose density gradients. Aliquots from each small fraction had been examined by immunoblotting with anti-MAL2 mAb 9D1, and antibodies to Compact disc59, used like a raft marker, also to TfR, a transmembrane proteins excluded from rafts. Fractions 1C4 represent the 40% sucrose coating and support Ambrisentan kinase activity assay the bulk of mobile membranes and cytosolic protein, whereas fractions 5C12 represent the 5C30% sucrose coating and support the rafts. The NH2-terminal peptide indicated in Fig. 1 A was selected to create a Ambrisentan kinase activity assay mAb to human being MAL2. The 9D1 hybridoma clone was defined as creating antibodies to MAL2 from the selective recognition of MAL2 in COS-7 cells transiently expressing c-MycCtagged MAL2 (MAL2-Myc) (discover Fig. 4 A) however, not in untransfected cells (Fig. 1 C). In keeping with the noticed expression from the gene (Fig. 1 B), mAb 9D1 known endogenous MAL2 in HepG2 and Caco-2 cells however, not in Jurkat or HeLa cells (Fig. 1 D). No cross-reactivity was noticed using the canine proteins in MDCK cells (unpublished data). Unlike observations of exogenous MAL2 in COS-7 cells, the endogenous MAL2 proteins in HepG2 and Caco-2 cells migrated as an assortment of glycosylated ( em M /em r = 30C40,000) and unglycosylated varieties ( em M /em r = 19,000). The observation that MAL2 glycosylation was delicate to treatment with endoglycosydase H (unpublished data) helps the usage of the initial consensus site of em N /em -glycosylation within the MAL2 molecule (Fig. 1 A). Fig. 1 E demonstrates endogenous MAL2 was detected Ambrisentan kinase activity assay in the raft small fraction of HepG2 cells selectively. As settings, we noticed that raft fractions included the GPI-anchored proteins Compact disc59 and excluded the transferrin (Tf) receptor (TfR), that have been used as reps of markers nonassociated and connected with lipid rafts, respectively. Open up in another window Shape 4. Depletion of endogenous MAL2 by transfection with an antisense phosphorothioate oligonucleotide. (A) The sequence of the antisense oligonucleotide used in MAL2 depletion experiments (MAL2/AS) and its alignment with wild-type MAL2 mRNA and the recombinant MAL2 mRNA species expressed in HepG2 cells are shown. Note that the changes introduced in the recombinant MAL2 transcripts prevent pairing with oligonucleotide MAL2/AS. The singly underlined residues correspond to sequences in the vector located immediately upstream of the inserted cDNA sequence. The doubly underlined residues in the coding sequence indicate nucleotides replaced by an equivalent triplet (MAL2-e), deleted.