Figure 1.
mCD300e is an N-glycosylated surface receptor. A, the phylogenetic tree of mouse LMIR3 (CLM-1/CD300f), LMIR4 (CLM-5), LMIR5 (CLM-7/CD300b), LMIR6 (CLM-2/CD300e), and LMIR7 (CLM-3) is shown on the basis of homology with the immunoglobulin-like domain (upper panel). The percentage of amino acid sequence identity of the immunoglobulin-like domain is indicated. Alignment of amino acid sequences for mCD300e is shown (lower panel). The putative signal sequence is shown in lowercase. An immunoglobulin-like domain is boxed. The transmembrane domain is underlined. The potential N-linked glycosylation site is shaded. The positively charged amino acid residue lysine within the transmembrane domain is shown in bold. B, Ba/F3 cells were transduced with FLAG-tagged mCD300e or mock. The transfectants were stained with mouse anti-FLAG Ab or mouse IgG1 Ab followed by PE-conjugated anti-mouse IgG goat F(ab′)2 Ab. C, lysates of Ba/F3 cells expressing FLAG-tagged mCD300e, mCD300e-N84Q, or mock were pretreated with or without N-glycosidase F before immunoprecipitation with mouse anti-FLAG Ab and subsequently immunoblotted with rabbit anti-FLAG Ab. D, 293T cells were transiently co-transfected with FLAG-tagged mCD300e construct or mock together with Myc-tagged FcRγ or DAP12 construct or mock. Immunoprecipitates of lysates of these transfectants with mouse anti-FLAG Ab were probed with anti-Myc Ab or rabbit anti-FLAG Ab. B–D, a representative of three independent experiments is shown. N-Gly, IB, or IP indicates N-glycosidase F, immunoblot, or immunoprecipitation, respectively.