Suppl. Figure 1: F604S FP mediated protein stabilization is a post-translational event.
Total cDNA was used to amplify FP with the primers mentioned for RT-PCR. GAPDH was
used as control.
Suppl. Figure 2: F604S FP half-life is more than FP. FP and F604S FP were transiently
expressed in HEK293 cells and treated with cycloheximide (40μg/ml) at indicated time
points. Cell lysates were then subjected to the indicated antibodies (A). To determine the FP
protein's half-life, cells expressing FP or F604S FP were labeled with 35S and chased for the
indicated time periods. Lysates were subjected to immunoprecipitation using anti-PDGFRA
antibody and eluted with 0.5% SDS by boiling for 5 min and analyzed by autoradiography
(B).
Suppl. Figure 3. SRC interacts with FP and the interaction is mediated via the SH2
domain of SRC. The SRC-SH2 domain was expressed as a GST-fusion protein and lysates
from cells expressing FP and F604S FP were incubated with SRC-SH2 domain. The bound
proteins were detected by immunoblotting. Equal protein loading was shown by amidoblack
staining.
Suppl. Figure 4: Kinase dead (G610R) FP failed to degrade the protein after SRC over
expression. HEK293 cells were co-transfected with G610R FP (KD) alone and together with
SRC. Cell lysates were subjected to western blotting with indicated antibodies.
Suppl. Figure 5: Pretreatment of SRC inhibitor (PD166326) rescues the degradation of
FP. FP and F604S FP were stably expressed in Ba/F3 cells, treated with (+) or without (-) the
SRC inhibitor PD166326 and the cell lysates were subjected to immunoblotting with
PDGFRA antibody.
Suppl. Figure 6: F604S FP and SHP-2 interaction is mediated by the phosphatase
domain of SHP-2. FP and F604S FP were expressed in HEK293 cells and in vitro binding
studies were carried out on the bacterially purified different domains of SHP2 (indicated in
the figure). The bound proteins were analyzed using SDS-PAGE followed by western
blotting.
Suppl. Figure 7: F604S FP interacts directly with the phosphatase domain of SHP-2. FP
and F604S FP were translated in vitro and binding studies carried out on the bacterially
purified GST-SHP2 phosphatase domain. The bound proteins were analyzed using SDSPAGE followed by western blotting.
Suppl. Figure 8: L629P FP stabilizes the protein. Ba/F3 cells expressing FP and L629P FP
were grown in the presence of IL-3 and cell lysates were prepared and analyzed for FP protein
levels.