Supplementary Material for Spatial Organization of Rho GTPase signaling by RhoGEF/RhoGAP proteins Paul M. Müller*, Juliane Rademacher*, Richard D. Bagshaw*, Keziban M. Alp, Girolamo Giudice, Louise E. Heinrich, Carolin Barth, Rebecca L. Eccles, Marta Sanchez-Castro, Lennart Brandenburg, Geraldine Mbamalu, Monika Tucholska, Lisa Spatt, Celina Wortmann, Maciej T. Czajkowski, Robert-William Welke, Sunqu Zhang, Vivian Nguyen, Trendelina Rrustemi, Philipp Trnka, Kiara Freitag, Brett Larsen, Oliver Popp, Philipp Mertins, Chris Bakal, Anne-Claude Gingras, Olivier Pertz, Frederick P. Roth, Karen Colwill, Tony Pawson, Evangelia Petsalaki+, + Oliver Rocks * these authors contributed equally + corresponding author: Email:
[email protected] (O.R.);
[email protected] (E.P.) Materials and Methods Mammalian cell culture Human embryonic kidney (HEK293T, ATCC number: CRL-3216) cells were cultured in Dulbecco's modified Eagle medium (DMEM) containing 10% fetal bovine serum (FBS) and 1% Penicillin/Streptomycin (37°C, 5% CO2) and transfected using polyethylenimine (PEI) dissolved in Opti-MEM. RhoGDI-knockdown HEK293T cells were generated by infection of cells for 24 h with lentiviral shRNA targeting RhoGDI and selection with puromycin (2 µg/ml). MDCK II canine kidney cells were cultured in modified Eagle medium (MEM) containing 10% FBS and 1% Penicillin/Streptomycin and transfected using Effectene (Qiagen). COS-7 cells were cultured in DMEM supplemented with 10% FBS and 1% Penicillin/Streptomycin (37°C, 5% CO2) and transfected using Lipofectamine 3000. The mCherry-Paxillin COS-7 reporter cell line was generated by infection of cells with a lentivirus encoding a Gateway system mCherry-Paxillin expression construct under the control of the Ubiquitin C (UbC) promoter (provided by Alexander Löwer, TU Darmstadt).