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D2f (8) or Schneider cells were incubated with medium containing sSpitz for 12 min. After incubation the cells were lysed with sample buffer, and lysates were separated on a 12% SDS-polyacrylamide gel electrophoresis (PAGE) gel. After blotting to nitrocellulose, the general antibody to ERK was detected with a 1/10,000 dilution of Ab. 7884, and dp-ERK was detected with a 1/2000 dilution of anti-dp-ERK. For induction of dp-ERK in embryos, embryos containing sev-hs-Gal4 and UAS-secreted Spitz4a were collected for 5 hours, administered heat shock at 37°C for 20 min, incubated at 29°C for 1 hour, and lysed. Control embryos of the same genotype were treated identically, but heat shock was eliminated.
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Cells were fixed in 4% formaldehyde (fresh) for 20 min. After washes, the cells were permeabilized with 0.5% Triton X-100 in phosphate-buffered saline for 5 min. Subsequent steps were standard. Fresh embryos were fixed in 8% formaldehyde and kept in 100% methanol at -20°C. All washes were done with PBS, 0.1% Tween-20. In cases of DAB or fluorescent double stainings, the second primary antibody was added only after completion of washes of the secondary antibody to dp-ERK to ensure efficient staining and avoid cross-reactivity. All other aspects of staining were standard. After preincubation of anti-dp-ERK with the phosphorylated peptide antigen, staining was eliminated. Other antibodies used include rabbit anti-β-Gal (Cappel). Secondary antibodies include horse-radish peroxidase-conjugated goat anti-mouse immunoglobulin G (IgG) and fluorescein isothiocyanate-or LRSC-conjugated goat anti-mouse IgG+IgM or anti-rabbit IgG (Jackson labs).
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Fixation of imaginal discs was carried out in 4% formaldehyde for 30 min. Wash solutions contained 0.2% Triton X-100. In contrast to anti-dp-ERK staining of embryos, which is highly reproducible, staining of imaginai discs is variable, for unknown reasons.
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We thank Y. Dolginov and D. Zharhary (Sigma Israel Chemicals, Rehovot, Israel) for help preparing activated antibody to MAPK (dp-ERK), and A. Brand, S. Crews, M. Freeman, W. Gehring, E. Hafen, C. Klämbt, M. Leptin, M. Levine, L. Perkins, and N. Perrimon for providing antibodies and fly stocks. We thank members of the Shilo lab, R. Schweitzer, and T. Volk for critical reading of the manuscript. R.S. is an incumbent of the Samuel and Isabela Friedman Career Development Chair. Supported by a grant from the Dr. Josef Cohn Minerva Center for Biomembrane Research (R.S.) and by grants from the Tobacco Research Council, US-Israel binational fund, UK-Israel binational fund, and Minerva Foundation (B.-Z.S.).
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