Fig. 2. Concentration-dependent Pelle phosphorylation in SL2 cells. (A) Pelle is autophosphorylated in SL2 cells when overexpressed. Increasing amounts of Pelle or PelleK240R expression vectors were transfected into SL2 cells. After 48 hours, cells were lysed, and subjected to anti-Pelle (A, top) or anti-Dorsal (A, bottom) immunoblots. The amount of cell lysate used was normalized by ß-Gal assays. Lane 1, vector; lanes 2-5: 0.03, 0.1, 0.3 or 1 µg of PelleK240R DNA was transfected. Lanes 6-9: 0.03, 0.1, 0.3 and 1 µg of Pelle DNA was transfected. (B) Pelle autophosphorylation occurs concomitantly with Dorsal activation. Cell lysates from the same transfections as above were used for CAT assays. Dorsal activation was quantitated by measuring expression from the co-transfected Dorsal-responsive CAT reporter. CAT activity is represented by the ratio of CAT/ß-Gal (see Materials and Methods).
