Supplemental Data The SCF/Slimb Ubiquitin Ligase Limits Centrosome Amplification through Degradation of SAK/PLK4

Constructs, RNAi and Cell lines Slimb Gateway® (Invitrogen) entry vector and pAc5-Slimb ∆ F box were kindly provided by Daniel Kalderon(1) and Isaac Edery(2), respectively. pAWH (HA) and pAWM (Myc) (Actin 5C promoter) destination vectors were acquired from the DRGC. A Gateway® (Invitrogen) destination vector was constructed by Luisa Capalbo (DMG) to express N-terminal-tagged ProteinA under the control of a methalothionein promoter (pMT-ProtA; this tag is composed of two IgG binding domains of the Staphylococcus aureus protein A). For the construction of the Non-Degradable SAK mutant (SAK-ND), two mutations were introduced in the DSGIIT degron in the SAK entry vector (Gateway). We used the Quick-Change XL Site-Directed mutagenesis kit from Stratagene. The mutations were as follows: the serine in position 294 and threonine in position 297 were mutated to alanine. Primers used for Site-Directed Mutagenesis are listed in Supplementary Information Table S1. pMT-GFP-SAK-ND and pMT-GFP-SAK constructs were made with the Gateway system from Invitrogen. pMT vectors were kindly provided by João Rocha. RNAi experiments, transfections and stable cell lines were performed as previously described(3). For induction of pMT - constructs, cells were incubated in medium containing 200μM CuSO4 for 15 hours, unless otherwise indicated. Primers used for dsRNA production from genomic or cDNA are listed in Supplementary Information Table S2.