Mouse anti ha

Antibodies used for western blot were: mouse anti-Flag (1:5,000, F3165, Sigma), mouse anti-Myc (1:1,000 sc-40, Santa Cruz), mouse anti-HA (1:1,000, sc-7392, Santa Cruz), mouse anti-GFP (1:500, sc-9996, Santa Cruz), mouse anti-α-tubulin (1:1,000, T5168, Sigma), rabbit anti-phospho-histone H3 (Ser 10) (1:5,000, 05-817 R, Millipore), rabbit anti-BubR1 (1:5000, A300-386A, BETHYL), mouse anti-Bub1 (1:1000, B0561, Sigma), rabbit anti-Bub3 (1:1000, ab131157, Abcam), rabbit anti-CENP-E (1:5,000, a gift from Dr. Don W. Cleveland), sheep anti-HOIP and anti-HOIL-1L (1:5,000, a gift from Dr. Philip Cohen), rabbit anti-SHARPIN (1:1,000, #4444, CST), anti-linear Ub^{62 (link)} (1:2500, a gift from Dr. Vishva M. Dixit, Genentech Inc.), rabbit anti-Ub K48 (1:1000, 05-1307, Millipore), rabbit anti-Ub K63 (1:1000, 05-1308, Millipore), rabbit anti-Ska3 (1:3000, a gift from Dr. Hongtao Yu), rabbit anti-KNL1 (1:5000, a gift from Dr. Hongtao Yu). Western blot antibody against SHARPIN of primary MEF cells was: rabbit anti-SHARPIN (1:500, Millipore). Images have been cropped for presentation (uncropped scans of the blots were shown in Supplementary Fig. 7).

HEK293T were transfected with equal amount of GFP-RIG-I and HA-Ub and with Myc-MEX3A in 1:20, 1:10 and 1:5 ratio compared to GFP-RIG-I amount. After 24 h from transfection, cells were treated with MG132 (50 µM) or DMSO as control, for 4 h. Cells were lysed in a solution containing RIPA buffer, as described above. Lysates were subjected to immunoprecipitation with mouse anti-RIG-I (Santa Cruz Biotechnology), overnight, at 4 °C, with rotation. The immunoprecipitated proteins were then washed five times with the RIPA lysis buffer, resuspended in sample loading buffer, boiled for 5 min, resolved in SDS-PAGE and then subjected to immunoblot analysis. Polyubiquitylated forms were detected by using mouse anti-HA from Santa Cruz Biotechnology.

MEFs were lysed with denaturing buffer (1% SDS, 50 mM Tris-HCl at pH 7.5, 0.5 mM EDTA, 1 mM DTT) to disrupt protein-protein interactions. Lysates were then diluted 10 times with NETN lysis buffer and subjected to immunoprecipitation with anti-βTrCP (Santa Cruz Biotechonology) overnight at 4 °C with rotation. The immunoprecipitated proteins were then washed five times with the NETN lysis buffer, resuspended in sample loading buffer, boiled for 5 min, resolved in SDS-PAGE and then subjected to immunoblot analysis. Polyubiquitylated forms were detected using mouse anti-HA from Santa Cruz Biotechnology.

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 cycG^HR7 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).

Drosophila S2 cells were cultured in Drosophila SFM (Invitrogen) with 10% fetal bovine serum, 100 U ml⁻¹ of penicillin and 100 mg ml⁻¹ of streptomycin at 24 °C. Transfections were carried out using the Calcium Phosphate Transfection Kit (Specialty Media). Immunoprecipitation and western blot analysis were carried out as previously described [33 (link)]. Antibodies used were as follows: mouse anti-Myc (Santa Cruz Biotechnology, Santa Cruz, CA, USA, sc-40); mouse anti-HA (Santa Cruz Biotechnology, sc-7392); mouse anti-Flag (Sigma, M2 F3165); rabbit anti-p-Hpo/Mst1/2 (Cell Signaling Technology, Danvers, MA, USA, #3681). The phospho-specific antibody against Wts-T1077 was a gift from Dr DJ Pan [34 (link)].