Figure 1.

Purification of rat epiregulin from the conditioned medium of AII-stimulated RASM cells. (A) Five liters of AII-stimulated RASM cell conditioned medium was concentrated and applied to an equilibrated anion-exchange column. Fractions (3 ml), eluted with a NaCl gradient (absorbance at 280 nm, ○), were assayed (50 μl/0.5 ml) for mitogenic activity in Swiss 3T3 cells (●). (B) Mitogenic fractions from A (nos. 8–16) were concentrated and applied to a gel exclusion column (absorbance at 280 nm, □) calibrated with protein standards (thyroglobulin and gamma globulin, 670 kDa and 158 kDa, respectively; ovalbumin, 44 kDa; myoglobin, 17 kDa; vitamin B₁₂, 1.35 kDa). Fractions (0.35 ml) were assayed for mitogenic activity (25 μl/0.5 ml) in Swiss 3T3 cells (■). Mitogenic fractions from two gel exclusion column runs (nos. 22–29, peak C) were concentrated and applied to a C₄ reverse-phase column. Recovered proteins were used for N-terminal amino acid sequencing. In a similar C₄ column run, selected fractions (nos. 19–23) were assayed for mitogenic activity (50 μl/0.5 ml) in Swiss 3T3 cells ( C). Remaining fractions were resolved by SDS/PAGE and proteins were visualized by silver staining (D).