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Fig. 7. Src42A/E-cad/Arm ternary complex formation and Src-dependent Arm
tyrosine-phosphorylation. (A) Arm/Src42A signals in E-cad immunoprecipitates
of untransfected and Src42A/arm-transfected S2 cells. (B)
Comparison of Arm/Src42A signals in cytosolic and membranous anti-Fas3 and
anti-Arm immunoprecipitates of wild-type embryos. Identical amounts of
membrane and cytosolic Arm were precipitated and examined. (C)
Immunoprecipitation of membranous and cytosolic fractions of wild-type,
Src42A[KR], Src42A[WT] and Src42A[YF] embryos. Same volumes of membrane and
cytosolic fractions were analyzed. (a-d) Western blots of fractionated
extracts. Tubulin is used as a control. (e-g) Arm, E-cad and Src42A blots of
Anti-Arm antibody immunoprecipitates. (h) Src42A blots of anti-E-cad-antibody
immunoprecipitates. (i,j) Arm blots of the supernatant of anti-Src42A antibody
immunoprecipitates with (j) or without (i) anti-pTyr-antibody treatment. (D)
Pull-down assay. Src42A was dissected as shown in the lower margin and
expressed as MBP fusions. Y indicates the autophosphorylation site.
GST-Arm-repeat signals were detected only in lanes 6 and 8, indicating Arm
repeats to bind to the 14 amino acid, autophosphorylation-site-containing
Src42A[Kinase] fragment. Lane 10 may indicate that the kinase domain other
than the autophosphorylation peptide is unrelated to Arm/Src42A interactions.
Input protein signals are shown in lower half as anti-MBP antibody signals.
(E) Src dependency of Arm tyrosine phosphorylation. Western blots of extracts
from S2 cells transfected with various combinations of metallothionein
(MT)-GAL4 and UAS-arm, and dsRNAs indicated are shown (a-f). Signals
for whole cell (d) and E-cad-free (e) tyrosine-phosphorylated Arm
significantly reduced by Src42A RNAi (compare lane 2 with lane 5).
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