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Fig. 5. Mop is required for MAPK phosphorylation and EGFR cleavage.
(A-C) Drosophila eye disc with mopT612
mutant clones marked by the absence of GFP (A, green in C) and stained with
anti-EGFR (B, magenta in C). EGFR levels are slightly increased in
mop mutant clones. (D) Western blot with Mop antibody of
extracts from S2 cells transfected with Actin-GAL4 and
UAS-mop, untreated (wt) or treated with mop dsRNA.
mop is expressed in S2 cells and its levels can be significantly
reduced by RNAi. (E) D2F cells were treated with lacZ or
mop dsRNA and incubated with sSpi-conditioned media for 0 or 30
minutes. Protein lysates were blotted with antibodies to diphospho-MAPK and
total MAPK. mop dsRNA treatment resulted in a decrease in MAPK
phosphorylation. (F) Western blot with anti-EGFR of lysates from D2F
cells treated with lacZ or mop dsRNA and incubated with
sSpi-conditioned media for the indicated times. Cells treated with
mop dsRNA failed to accumulate a faster-migrating band recognized by
the EGFR antibody (asterisk). The position of full-length EGFR is indicated by
an arrow. (G) S2R+ cells were treated with lacZ, mop, Cbl
and/or sty dsRNA as indicated, and transfected with
Actin-GAL4, UAS-GFP and UAS-EGFR
top, and
UAS-CblL in the indicated lanes. Protein lysates were blotted with
antibodies to EGFR and GFP (transfection control). A smaller band recognized
by the EGFR antibody that is the same size as the smaller band in F, is
indicated by an asterisk; the arrow indicates full-length EGFRtop. The
proportion of the smaller band is increased by Cbl cotransfection or
sty RNAi and decreased by mop RNAi. The lower three panels
show RT-PCR quantification of mop, Cbl and sty mRNA,
demonstrating the efficiency of the RNAi treatment.
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