Supplemental Figure 1

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1 Supplemental Fig. 1. Kinetics of,,, AKT and ERK activation in BMMCs following SCF stimulation. Starved BMMCs were stimulated with 250ng/mL of SCF for the indicated time. Soluble Cell Lysates (SCLs) were analysed by western-blotting (60µg of proteins per lane). KIT,,,, AKT and ERK activation were assessed using specific phospho-antibody and each membrane was then stripped and reprobed with their respective antibodies. The last lane was loaded with a lysate of BMMCs obtained from KIT-D816V transgenic mice (BMMC16M). Supplemental Fig. 2. Effect of jak3 sirna on STAT phosphorylation. P815 cells, treated either with control (c) or jak3 sirna, were starved and lysed. The SCLs were immunoblotted with phopho-stat specific antibodies as indicated and anti-erk as loading control. Supplemental Fig. 3. GSK3, p38, PKA, PKG, and CAMKII are not implicated in, -3, - 5 serine phosphorylation. P815 and HMC-1 cells were starved overnight and subsequently treated with the indicated chemical inhibitors. The SCLs were immunoprecipitated using anti-stat specific antibodies. Immunocomplexes were resolved by SDS-PAGE and probed with anti-phosphoserine specific antibodies, stripped and reprobed with the corresponding anti-stat antibodies. (A) H-89 is a PKA inhibitor, PKG is a PKG inhibitor and SB is a GSK3 inhibitor. (B) SB is a p38 inhibitor. (C) KN-93 is a CAMKII inhibitor. (D) SB216763, SB and U0126 were controlled on their respective molecular targets in both HMC-1 and P815 cells. The inhibitors did not show activity on KIT tyrosine phosphorylation. Supplemental Fig. 4. Controls for the interaction between KIT and STAT proteins. (A) Interaction of KIT and in HMC-1 cells. Co-immunoprecipitations were done as in Fig. 4. Lysates were immunoprecipitated either with anti-kit or control IgG1 (c) antibodies. Dasatinib was used at 1 µm. (B) Co-immunoprecipitations of STAT proteins with KIT were done as in Fig. 4. The lysate (SCL) is shown to compare protein expression in the lysate with the bound fraction. The panel showing was deliberately overexposed to show some remaining KIT phosphorylation. Supplemental Fig. 5. Peptide pull down assays using KIT and control peptides. HMC-1 lysate was used for affinity pull-down assay with 2 nmol of either non phosphorylated peptide (Y568Y570), or mono-phosphorylated peptides (py568 and py570) or bi-phosphorylated peptide (py568py570). Non phosphorylated (YXXM) and phosphorylated (pyxxm) CD28 derived peptides were used to control the binding specificity. Bound and were revealed with specific antibodies. Lysate (SCL) was used as positive control. Anti-phosphotyrosine (4G10) was used to control the phosphorylation state of the peptides.

2 Supplemental Figure 1 SCF (min/h) WT BMMC h 24h BMMC 16M py701- py705- py694- pt202/y204-erk ps473-akt AKT

3 Supplemental Figure 2 P815 sirna C JAK3 JAK3 py701- py705- py694- ERK2

4 Supplemental Figure 3 A IP: pser727- IP: pser727- IP: pser726/731- H-89 (2 µm) : PKG Inhibitor (2 µm) : SB (2 µm) : P815

5 Supplemental Figure 3 B C pser727- pser727- pser726/731- IP: IP: IP: pser727- pser727- pser726/731- IP: IP: IP: SB (2 µm) : P815 HMC-1 KN-93 (2 µm) : P815 HMC-1

6 Supplemental Figure 3 D HMC-1 P815 SB (2 µm) : ps9-gsk3 GSK3 HMC-1 P815 SB (2 µm) : pt180/y182-p38 p38 HMC-1 P815 U0126 (2 µm) : pt202/y204-erk ERK2

7 Supplemental Figure 4 A IP B IP: KIT SCL KIT C KIT C SCL - + Dasatinib (1 µm) Dasatinib KIT KIT

8 4G10 pyxxm YXXM SCL py568py570 py568 py570 Y568Y570 Supplemental Figure 5