Supporting Information

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Generation of KIT-Expressing BaF3 Cells. The retroviral vector pMSCV (36) containing a 5' LTR-driven murine KIT internal ribosome entry site (IRES) enhanced GFP was used to generate high-titer helper-free retrovirus stocks prepared by transient cotransfection of 293T cells (1). Generation of KIT-expressing BaF3 cells was similar to that described for the generation of BCR-ABL expressing BaF3 cells (2).

KIT Immunoprecipation. KIT expressing BAF/3 cells were set up at 5 × 10⁶ cells per 10-cm plate in the presence of 100 ng/ml stem cell factor (SCF). NaPP1[4-amino-1-tert-butyl-3-(1-naphthyl)pyrazolo[3,4-D]pyrimidine] (2 mM), imatinib mesylate (2 mM), and ACK45 (10 m g/ml) were added to cultures for 2 h. An equivalent volume of DMSO was added to control cultures. Cells were lysed in 500 m l of detergent lysis buffer (3) and 5 m g of KIT antibody (BD Pharmingen catalog no. 553352) was added for 4 h. Five micrograms of rabbit anti-rat IgG (DAKO catalog no. 3046) was added, and precipitation was continued overnight. Proteins A (Amersham Pharmacia catalog no. 170974-01) and G (Amersham Pharmacia catalog no. 170618-01) were added to samples and incubated for 60 min with mixing. Precipitates were washed three times and resuspended in sample buffer and boiled for 1 min. A total of 10⁶ cell equivalents were run on gel and Western blotted for KIT (catalog no. 06-438, Upstate Biotechnology, Lake Placid, NY) and phosphotyrosine (Upstate Biotechnology catalog no. 05-321).

1. Sherr, C. J. (1996) Science 274, 1672-1677.

2. Sherr, C. J. (2004) Cell 116, 235-46.

3. Pear, W. (1996) in Current Protocols in Molecular Biology, eds. Ausubel, F. M., Brent, R., Kingston, R. E., Moore, D. D., Seidman, J. G., Smith, J. A. & Struhl, K. (Wiley, Boston), Vol. 2, pp. 9.11.1-911.18.

Fig. 6. NaPP1 [4-amino-1-tert-butyl-3-(1-naphthyl)pyrazolo[3,4-D]pyrimidine] does not suppress stem cell factor (SCF)-induced KIT tyrosine phosphorylation and cytokine-independent growth, unlike ACK45 and imatinib mesylate. (A) NaPP1 (2 m M), imatinib mesylate (2 m M), and ACK45 (10 mg/ml) were added to KIT-expressing BaF3 cells in the presence of 50 ng/ml SCF. KIT was immunoprecipitated from cell lysates 6 h after inhibitor addition, and levels of KIT and KIT tyrosine phosphorylation were measured as described in Supporting Text. (B) NaPP1 (2 mM), imatinib mesylate (2 m M), and ACK45 (10 mg/ml) were added to KIT-expressing BaF3 cells in the presence of 50 ng/ml SCF every 24 h, and cell counts as measured by trypan blue exclusion were obtained 48 h after inhibitor addition. Two separate experiments were conducted with similar results.