Tandem affinity purification of GLI1 complexes

The epitope-tagging strategy to isolate GLI1-containing protein complexes from human cells was performed essentially as described previously⁵⁵. HEK293 cells infected with HA-FLAG-Gli1 recombinant retrovirus were grown in 293SFM medium and harvested at near confluency (~1 × 10⁹ cells). The cells were separated into cytoplasmic and nuclear fractions. Cytoplasmic fraction was dialysed for 5 h in a buffer consisting of 20 mM Hepes–KOH (pH 7.9), 20% glycerol, 0.1 M KCl, 0.2 mM EDTA, 0.5 mM PMSF, and 0.5 mM DTT. The lysate was centrifuged at 15,000×g for 15 min at 4 °C. The supernatants were immunoprecipitated with anti-FLAG antibody-conjugated M2 agarose (400 μl; Sigma-Aldrich). Bound proteins eluted 0.4 mg ml⁻¹ FLAG peptide (Sigma-Aldrich) were further affinity purified using 20 μl anti-HA antibody-conjugated agarose (Roche Diagnostics). The final elutes from HA beads eluted with 2.5 mg ml⁻¹ HA peptide (Roche Diagnostics) were separated by SDS–PAGE on a 5–20% gradient gel for silver staining analysis. Specific bands were excised from the gel and subjected to peptide sequencing by mass spectrometry using a 4700 proteomics analyser (AB SciEx). Data were analysed by the Mascot search engine (Matrix Science).