Anti gsk3β

The SCF components such as human influenza hemagglutinin (HA)-Skp1, Cul1, Myc-Rbx1, and FLAG-Fbxo7 or FLAG-Fbxo7(ΔF-box) were transfected in HEK293T cells by using polyethylenimine. After 48 h of transfection, the cells were harvested and resuspended in lysis buffer (LB) (25 mM Tris–HCl, pH 7.5, 225 mM KCl and 1% NP-40) containing a protease inhibitor cocktail (Sigma-Aldrich, St. Louis, MO) and phosphatase inhibitors (10 mM NaF and 1 mM Na₃VO₄). The lysates were incubated with agarose anti-FLAG M2 beads (Sigma-Aldrich, St. Louis, MO) for 6 h at 4°C with rocking. Beads were washed with LB and the SCF complexes eluted with FLAG elution buffer (300 µg/ml of peptide FLAG in 10 mM HEPES pH 7.9, 225 mM KCl, 1.5 mM MgCl₂, and 0.1% NP-40) for 1 h at 4°C with rocking. The eluates were stored in 15% glycerol at −20°C until use. To evaluate the purification of SCF complexes, immunoblotting was performed and probed using anti-Fbxo7 (ABN1038, Merck Millipore, Watford, UK), anti-HA (Abcam, Cambridge, UK), anti-Gsk3β (Santa Cruz Biotechnologies, CA, USA), anti-Tomm20 (Abcam, Cambridge, UK), or anti-myc (Cell Signaling Technologies, MA, USA). The concentration of the complexes was determined against known concentrations of BSA by Coomassie blue staining of the gel. The densitometry of the bands was determined by ImageJ.