SDS-PAGE and Western blot analysis of DCH and variants. DCH (lane 2), DCH-mu (lane 3), and SCH (lane 4) were expressed in CHO cells and purified by Q-sepharose anion exchange chromatography, immobilized metal ion affinity chromatography, and gel filtration. (A) Purified proteins were analyzed on 12% SDS-PAGE under reducing conditions. (B) Proteins were blotted onto nitrocellulose membranes. HD70-GM-CSF (solid arrowhead) and HD70-mu-GMCSF (solid arrowhead) polypeptides were detected with an anti-penta-His mouse IgG1 Ab in combination with an alkaline phosphatase-conjugated goat anti-mouse Ab. The HD70-IL-2 (open arrowhead), HD70-mu-IL-2 (open arrowhead), and HD70-C kappa (open arrowhead) polypeptides were detected using an alkaline phosphatase-conjugated anti-human kappa light chain Ab. Molecular weight markers and their molecular weights in kilodaltons are shown in lanes 1 (Multi Mark multicolored standard) and 5 (Mark 12 unstained standard).