FIGURE 3.

STAT1 activation upon treatment with IFNα-2a-EGF chimeric activators. Starved HeLa (A), A431 (B), Daudi (C), or Daudi-EGFR (D) cells were incubated for 30 min with phosphate-buffered saline (vehicle), commercial IFNα A, EGF, and IFNα·EGF chimeric activator proteins containing wild-type or mutant IFNα-2a (CA-WT CA-K133A, CA-R144A, or CA-R149A) or the corresponding wild-type or mutant IFNα-2a proteins produced from Pichia (see “Experimental Procedures”). Lysates were prepared, and immunoblots were performed as described under “Experimental Procedures” probing with an anti-STAT1(pTyr⁷⁰¹) antibody and with an anti-actin antibody as a loading control. (See also supplemental Fig. 1, which shows similar immunoblots in which lanes were scanned with a densitometer and the phospho-STAT1 signal was normalized to the actin signal.) HeLa, A431, and Daudi-EGFR cells express both EGFR and IFNAR, whereas Daudi cells express only IFNAR. rhIFNαA stands for recombinant human interferon α A.