Full length wdb was PCR amplified from cDNA (LD34343, obtained from DGRC, Bloomington IN, USA) and cloned as BglII/NotI fragment into pEG and VP16 (BamHI/NotI) vectors. pJG-CycG (1–566), GST-CycG (1–215) and GST-CycG (215–566) DNA was a gift from F. Peronnet, France [46 (link)]. The CycG subdivision constructs were PCR-amplified using the pJG/GST-CycG constructs as template and further subcloned as EcoRI/XhoI fragments in either pEG, pJG or VP16 vectors [59 (link)], [60 (link)]. Protein-protein interaction assays were done according to standard protocols using the Brent two-hybrid system [59 (link)]. Protein expression in yeast cells (EGY40: Mata, ura3, his3, trp1, leu2, GAL) was verified either with mouse anti-HA (1:1000; St. Louis MO, USA), mouse anti-VP16 (1:100; Santa Cruz Biotechnology, Dallas, USA) or rabbit anti-LexA antibodies (1:1000; Bio Acadamia, Osaka, Japan).
CycG or Wdb protein was immuno-precipitated from about 500 embryos (0–24h) using either anti-CycG antibodies or anti-Wdb antibodies (see supporting materials and methods) as described before [46 (link)]. Akt1 and Wdb complexes were co-immunoprecipitated from 150 heads each of either wild type or cycGHR7 homozygous mutant animals using rabbit anti-Akt1 (1:50; Cell Signaling Technology; Danvers MA, USA) and detected with rabbit anti-Akt1 or rat anti-Wdb (see supporting materials and methods).
CycG or Wdb protein was immuno-precipitated from about 500 embryos (0–24h) using either anti-CycG antibodies or anti-Wdb antibodies (see supporting materials and methods) as described before [46 (link)]. Akt1 and Wdb complexes were co-immunoprecipitated from 150 heads each of either wild type or cycGHR7 homozygous mutant animals using rabbit anti-Akt1 (1:50; Cell Signaling Technology; Danvers MA, USA) and detected with rabbit anti-Akt1 or rat anti-Wdb (see supporting materials and methods).
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