Supplemental Figure 1
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Fig. S1. GST-APRO4
is not phosphorylated by Src. Briefly, purified GST-APRO4 or GST control
proteins were added to a constant amount (5 units) of purified active Src
kinase as described in the Material and Methods section except that [g32P] ATP and enolase were omitted
from the reaction. Proteins were then resolved by SDS-PAGE followed by
immunoblotting on a PVDF membrane. The membrane was then immunoblotted with
anti-phosphotyrosine monoclonal antibody (4G10) and then stripped before being
re-probed with anti-GST polyclonal antibody. Src protein was detected with the
anti-phosphotyrosine antibody whereas GST-APRO4 was not thereby indicating that
GST-APRO4 was not phosphorylated by Src. Note that Src tyrosine phosphorylation
was reduced in GST-APRO4 reaction in comparison to the GST control, which is
consistent with the fact that GST-APRO4 inhibits Src kinase activity.
Supplemental Figure 2
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Fig. S2.
Inhibition of Src Y529F and Fyn Y531F kinase activities in vitro by GST-APRO4.
(A,B) PC12 cells were transfected with either the constitutive active form of
Src (Src Y529F) or the constitutive active form of Fyn (myc-tagged-Fyn Y531F).
Anti-Src or anti-myc immunoprecipitates from transfected cells were assayed for
in vitro kinase activity in the presence of 5 µM of purified GST-APRO4 or 5 µM
of purified GST control proteins. Src and Fyn activities were measured in the
presence of [g32P]
ATP and enolase. Both Src Y529F and Fyn Y531F kinase activities were inhibited
by GST-APRO4 but not by GST alone. Immunoblotting with Src or myc antibodies
confirmed that equivalent amounts of Src or myc-Fyn were present in
immunoprecipitates reactions. Coomassie staining of SDS-PAGE of purified
GST-fusion proteins is shown.
