Figure 1.
Murine MICL is structurally and functionally similar to the human receptor. (A) ClustalW alignment of human and mouse amino acid sequences (GenBank accession numbers: NP_612210 and NP_808354). The ITIM sequences are in bold face and boxed, the transmembrane regions are underlined and the beginning of the CRD is indicated by arrow. The six conserved canonical cysteine residues of the CRD are in bold and underlined, while the two cysteines in stalk region, potentially involved in dimerisation, are ringed. Predicted N-glycosylation sites are highlighted in grey. “*”, identity; “:”, conservative substitutions; “.” similar substitutions. (B) Cartoon representations of hMICL and mMICL. (C) Flow cytometric analysis demonstrating surface expression of mMICL-HA in transduced NIH-3T3 fibroblasts and RAW264.7 macrophages, as indicated. Anti-HA staining (bold line) and isotype control (grey filled histograms). (D) Anti-HA Western blot analysis of murine or human MICL-HA transduced NIH-3T3 cell extracts, under non-reducing (upper panels) or reducing (lower panels) conditions. Cell extracts were incubated with PNGase, as indicated. (E) Anti-phosphatase Western blotting analysis of immunoprecipitates prepared from mMICL-HA transduced RAW264.7 macrophages, stimulated with pervanadate for the times indicated. These data show representative results from at least three independent experiments.