FIG. 3.

Presence and phosphorylated state of EGFR in NIKS cells. (A) Levels of P-EFGR (as detected by immunoprecipitation and Western analysis; see Materials and Methods) in NIKS cells either grown on fibroblast feeders in normal F medium containing 5% FBS and EGF (10 ng/ml) (lane 1), serum-starved for 24 h in F medium containing 0.1% FBS (lane 2), or serum-starved in F medium containing 0.1% FBS for 24 h followed by the addition of EGF (10 ng/ml) for 15 min (lane 3). Note the induction of P-EGFR in serum-starved, EGF stimulated cells. Indicated by the arrows at left is the migration position of P-EGFR and immunoglobulin (IgG) from the immunoprecipitation using anti-EGFR antibody (Ab-15; Labvision). The blot was probed with an antiphosphotyrosine antibody (PY20; Santa Cruz). (B) Levels of total EGFR protein (as detected by direct Western analysis using anti-EGFR antibody) (Ab-15; Labvision) in untransfected NIKS (N); three independently derived, transfected populations of NIKS cells harboring the WT HPV16 genome (W1, W2, and W3); three independently derived, transfected populations of NIKS cells harboring the E5^XCM− mutant HPV16 genome (M1, M2, and M3); and W12E cells (W12) grown in the absence (−) or presence (+) of EGF (10 ng/ml). Note the similar range in levels of EGFR in different populations of NIKS cells harboring the WT or E5^XCM− mutant HPV16 genomes and the similar modest reduction in levels of EGFR in cells grown in the presence of EGF at 10 ng/ml.