Leupeptin

The protease inhibitor cocktail was purchased from BioVision (Mountain View, CA, USA). Deuterium oxide was purchased from Cambridge Isotope Laboratories (Tewksbury, MA, USA). Chelating sepharose resin for Ni-IDA purification was purchased from Cytiva (Uppsala, Sweden). EDTA, Tris, and NaH₂PO₄ were purchased from Duchefa Biochemie BV (Haarlem, The Netherlands). HEPES, IPTG, leupeptin, lysozyme, kanamycin, and TCEP were purchased from Goldbio (St. Louis, MO, USA). Acetonitrile, H₂O, and MeOH were purchased from JT Baker (Phillipsburg, NJ, USA). DNase I was purchased from Roche (Basel, Switzerland). BODIPY-FL-GTP and BODIPY-FL-GTPγS were purchased from Thermo Fisher Scientific (Waltham, MA, USA). Acetic acid, benzamidine, formic acid, GDP, phosphoric acid, imidazole, and MgCl₂ were purchased from Sigma–Aldrich (St. Louis, MO, USA).

Unless otherwise indicated, all chemical reagents were obtained from Sigma or Fisher Scientific. Fluorescein di-β-D-galactopyranoside (FDG) was from Marker Gene Technologies, and the protein inhibitor mixture was from Sigma (catalog no. S8830). Leupeptin, pepstatin, and aprotinin were from Gold Biotechnology. All restriction endonucleases and E. coli strain BL21(DE3) were from Thermo Scientific. E. coli strain DH5α was purchased from New England Biolabs. Pfu ultra DNA polymerase used for site-directed mutagenesis was purchased from Agilent. [γ-³²P]GTP was from Perkin Elmer. Mouse monoclonal antibodies raised against α-tubulin (T6074), FLAG tag (F1804) or His-tag (H1029) were from Sigma. Mouse monoclonal antibody raised against hemagglutinin (HA) tag (clone 12CA5, #11583816001) was obtained from Roche. Mouse monoclonal antibody raised against MYC-tag (9B11, #2276) was from Cell Signaling. Rabbit polyclonal antibody raised against Gαs (C-18, sc-383) was purchased from Santa Cruz Biotechnology. Goat anti-rabbit Alexa Fluor 680 (A21077) and goat anti-mouse IRDye 800 (#926–32210) secondary antibodies were from Life technologies and LiCor, respectively.