Fig. 4.

Insertion of isoleucine into polyL25 restores activity. (A) Sequences of add-back mutants in which isoleucines were inserted at the indicated positions in polyL25. (B) Traptamers listed in A were tested for focus forming activity as in Fig. 1E. The results show the average of three independent experiments with SE indicated. (C) C127 cells were infected with high-titer stocks of empty MSCV vector or MSCV expressing the E5 protein or a polyL25 mutant containing a single isoleucine at the indicated position. Photomicrographs were taken at 100× magnification 10 d later. The titer of all virus stocks was similar. (D) Extracts were prepared from C127 cells harboring empty MSCV or MSCV expressing the E5 protein, LI-11, polyL25-I8,13,22, polyL25-I13, or polyL25 (polyL). Extracts were immunoprecipitated with anti-PDGFβR antibody, subjected to SDS/PAGE, and immunoblotted with antibody recognizing phosphotyrosine (Upper) or PDGFβR (Lower). (E) Extracts were prepared from serum-starved human diploid fibroblasts (MSCV) or cells stably transformed by polyL25-I18,13,22, or polyL25-I13. phospho-RTK arrays were processed as described in Fig. 2B. The tyrosine phosphorylated PDGFβR spots are underlined in red. (F) C127 cells were infected with low-titer retrovirus stocks of MSCV expressing BPV E5 or a FLAG-tagged polyL25 add-back clone containing isoleucine at the indicated position. Foci were counted 2 to 3 wk after infection, normalized for virus titer, and expressed relative to the activity of E5. Circles and triangles show independent experiments. Each symbol is the average of two plates of foci from a single infection. (G) C127 cells were infected with MSCV vector or MSCV expressing FLAG-tagged LI-5, polyL25, or a polyL25 clone with isoleucine inserted at the indicated position. After selection of puromycin-resistant cells, traptamer expression was determined by immunoprecipitation and immunoblotting with anti-FLAG antibody.