Figure 3.
Involvement of BACE1 in ST6Gal I secretion. (A) COS cells expressing rat ST6Gal I in the pSVL vector and either human BACE1 or vector were pulse labeled and chased for 3 h for further immunoprecipitation analysis by using anti-ST6Gal I antibody (C, cell lysates; M, media.) The percentage of secretion (mean ± SD, n = 3) was defined as the ratio of the radioactivity of soluble ST6Gal I in the medium to the total ST6Gal I radioactivity (medium + cells). (B) COS cells were transiently cotransfected with ST6Gal I FLAG-pSVL and either human BACE1 or vector. After 24 h of expression, soluble ST6Gal I FLAG in the media were pulled down with M2-agarose and then detected with an E41 antibody. (C) Cells expressing ST6Gal I plus either APPSW, C99 or control pcDNA3.1 vector, were pulse labeled and chased for 6 h, and analyzed as shown in A. The percentage of secretion, taken as an average of two independent experiments, was defined as the ratio of the radioactivity of soluble ST6Gal I in the medium to the total ST6Gal I radioactivity (medium + cells). APP and APPsβ were detected with 6E10 and βNL antibodies, respectively. (D) Microsome fractions were prepared from HEK293 cells that stably express BACE1 or from their parent cells, and equivalent amounts of proteins were used for staining with SNA lectin that recognizes Siaα2,6Gal oligosaccharide structure. (For SNA lectin staining, 40 μg of protein was used for lanes 1 and 2, 10 μg for lanes 3 and 4, and 20 μg for Coomassie staining.)