Fig. 3. Analysis of the expression and release properties of Agno deletion mutants by immunoprecipitation followed by Western blot.

(A) Analysis of the expression profiles of either WT Agno or its deletion mutants by immunoprecipitation/Western blot (IP/WB). Either Agno WT or its various deletion mutants were cloned into pCGT7 vector and transfected into 293HEKT cells as described in Materials and Methods. Whole-cells extracts (WCE) (200 µg) prepared from untransfected (lane 1) transfected (lanes 2–6) cells at 24 h posttransfection were subjected to immunoprecipitation (IP) using anti-T7 antibody followed by Western blot (WB) using the same antibody as described in Materials and Methods. (B) Analysis of the release properties of Agno WT and its deletion mutants by IP/WB. In parallel to the IP/WB analysis described for panel A, supernatants collected from each sample (200 µl) were subjected to IP/WB analysis using anti-T7 antibody as described in the Materials and Methods. Sup.: Supernatant, -Cont.: Control for the whole cells extracts prepared from the untransfected cells and for the supernatant collected from the untransfected HEK293T cells. A star sign and an arrow head for each panel point to the migration position of IgG light and IgG heavy chains respectively originating from the anti-T7 antibody that was used for IP. Transf.: Transfection., Mut.: Mutant.