Mcherry pak3 60 113 s74a f84a mcherry c1

All the plasmids used were previously developed and described in publication and are available on Addgene^{14 (link),19 (link),67 (link)}. Briefly, the Rac1 sensor consisted of mEGFP-Rac1 (Addgene #83950) and mCherry-PAK2 binding domain-mCherry (Addgene #83951), the Ras sensor consisted of pCI-mEGFP-HRas ((Addgene #18666) and pCI-mRFP-RBD^K65E,K108A- mRFP (Addgene #45149), the Cdc42 sensor consisted of mEGFP-Cdc42 (Addgene #29673) and mCherry-Pak3(60-113)/S74A/F84A-mCherry-C1 (Addgene #29676). PKCα (Bos taurus) was cloned into CMV-promoter-containing mEGFP C1 vectors such that mEGFP was fused to the N terminus of PKCα^{14 (link)}. mEGFP-PKCα∆QSAV was made by introducing a single point mutation into mEGFP-PKCα/C1 to introduce a stop codon before the last four amino acids (QSAV) of PKCα^{14 (link)}.

NG108-15 neuroblastoma cells were purchased from Sigma Aldrich and grown in Dulbecco's modified Eagle's medium (DMEM) with 10% (v/v) fetal bovine serum (FBS) (Invitrogen), 100 μg/ml streptomycin and 100 units/ml in penicillin in a 5% CO₂atmosphere at 37°C. For live cell imaging studies, cells were seeded on glass coverslips, coated for 3 h with laminin (50 μg/ml, L2020 Sigma) in 12-multiwell plates. Cells were transfected 24 h later with intermolecular RhoA/Cdc42 FRET sensors (Murakoshi et al., 2011 (link)) using Metafectene reagent (Biontex Laboratories) following the manufacturer's protocol and imaged 1 day after transfection. mEGFP-RhoA-C1 (Addgene plasmid #29674), mEGFP-Cdc42-C1 (Addgene plasmid #29673), mCherry-Rhotekin(8–89)-mCherry-C1 (Addgene plasmid #29675) and mCherry-Pak3(60–113)/S74A/F84A-mCherry-C1 (Addgene plasmid #29676) were a gift from Ryohei Yasuda (Murakoshi et al., 2011 (link)).