Fig. 2.

Secreted Ad19a E3/49K protein has a long half-life in the supernatant of Ad19a-infected cells and cells stably expressing E3/49K, and can be purified to homogeneity. (A and B) A549 cells infected with Ad19a for 9 h (A) and A549 cells stably expressing E3/49K (B) were metabolically labeled for 1 h with [³⁵S]-methionine and then chased with medium containing nonradioactive methionine for the indicated times. E3/49K was precipitated from lysates using the antiserum directed to the C terminus, whereas sec49K was precipitated with serum R48 against the N-terminal domain. (C) Supernatants of A549 cells stably transfected with the E3/49K gene were collected over a period of 1–9 d after placement in medium without FCS. Cellular debris was removed by centrifugation, and 15 µL of supernatant was directly analyzed by Western blot analysis using antiserum R48. (D and E) Sec49K was purified using affinity chromatography with mAb 4D1 coupled to protein G Sepharose beads. Sec49K was eluted from the columns collecting 500-µL fractions. Then 15 µL of each fraction was analyzed by SDS-PAGE, followed by Western blot analysis with antiserum R48 (D) or silver staining (E).