Anti cul3

The small fraction of tissue lysates prepared for mass-spectrometry experiments were used for western blotting. On average 10–15 μg of total protein from WT and Cul3^+/− cerebral cortex, cerebellum or hippocampus were resolved by SDS-PAGE and transferred onto PVDF Immobilon-P membranes (Millipore). After blocking with 5% nonfat dry milk in 1× TBS with 0.1% Tween-20 (TBST) for 1 h at room temperature, membranes were first probed overnight with the appropriate primary antibodies in 3% BSA in TBST, and then after 1 h of incubation with corresponding host specific HRP-conjugated secondary antibody (Abcam). Membranes were developed using the EZ-ECL chemiluminescence detection kit (Denville Scientific). The following primary antibodies were used: anti-Cul3 (1:1000; Cell Signaling), anti-RhoA (1:1000; Cell Signaling), and anti-Gapdh (1:5000; Sigma Aldrich) as a loading control. Quantification was performed by densitometry with ImageJ software. Western Blot images for quantification of Cul3, total RhoA and active RhoA are shown in the Supplementary Fig. S18.

Total cell extracts were prepared using Pierce IP lysis buffer (Thermo Fisher Scientific, USA) according to the manufacturer’s instructions. Equal amounts of proteins were subjected to WB analysis. The antibodies used for WBs were anti-β-Actin (mouse, Sigma, USA), anti-CUL1 (rabbit, Thermo Fisher Scientific, USA), anti-CUL2 (mouse, Santa Cruz Biotechnology, USA), anti-CUL3 (rabbit, Cell Signaling Technology, USA), anti-CUL4A (mouse, Sigma, USA), anti-CUL4B (mouse, Sigma, USA), anti-CUL5 (rabbit, Abcam, USA), anti-CUL7 (rabbit, OriGene, USA), anti-Flag (mouse, Sigma, USA), anti-RBX1 (rabbit, Cell Signaling Technology, USA), anti-DCAF1 (rabbit, Abcam, USA), anti-DCAF4 (rabbit, Sigma, USA), anti-DCAF8 (rabbit, Sigma, USA), anti-DCAF11 (rabbit, Sigma, USA), anti-DCAF15 (rabbit, Sigma, USA), anti-DDB1 (rabbit, Sigma, USA), anti-p21 (rabbit, Sigma, USA), and anti-p27 (mouse, Abcam, USA). Signals from WBs were recorded using a ChemiDoc MP (Bio-Rad, USA). All experiments were performed in triplicate.