Fig. 1. Time course of Spitz induction of Egfr signaling in S2 cells. (A) Protocol used in B,C, see text. (B) Blot probed for Egfr. Second, non-specific band (asterisk) serves as a loading control. The DNA used and the times after Spitz induction are indicated above. (C) Blot for pMAPK shows pathway activation (same gel as is shown in B); asterisk indicates the level of pMAPK activation after 5 minutes. Note, the pMAPK intensity at 0 minutes (no time with Spitz induction) is indistinguishable from that of the untransfected controls. Also note, there is no detectable pMAPK (above controls) in the Egfr^tsla transfections, whereas there is detectable Egfr antigen. (D) Protocol used in E-H, see text. Lysates were prepared by immunoprecipitation using anti-Egfr. (E,F) Blot probed for phospho-Tyrosine (`pTyr'). Note that, at 18°C (E), the anti-Egfr precipitable pTyr signal is detectable over control (black asterisk) for the Egfr⁺ and Egfr^tsla transfections (white asterisks), whereas at 30°C (F), Egfr⁺ yields detectable anti-Egfr precipitable pTyr signal (white asterisk), but Egfr^tsla does not (black asterisk). (G,H) The same gels as in E,F, re-probed for Egfr antigen.