Direct ip kit

Plasmids encoding Arrb2-Flag, Dvl2-GFP, and hemagglutinin (HA)-tagged Gβ and Gγ were transiently transfected into HEK293T cells. Bait proteins were affinity isolated on anti-Flag beads (Sigma-Aldrich, Munich, Germany) or on anti-GFP beads prepared by coupling an anti-GFP antibody (Acris Antibodies, Herford, Germany) covalently to agarose beads (Direct IP Kit; Thermo Fisher Scientific, Bremen, Germany). Eluates were digested with trypsin and subsequently with Lys-C in solution. After addition of protein digests as quantitative standards, the samples were desalted on C-18 stage tips (Nest Group, Southborough, MA) and analyzed by nanoflow high-performance liquid chromatography–tandem mass spectrometry (2D-NanoLC; Eksigent, Dublin, CA; Orbitrap Velos MS, Thermo Fisher Scientific; Vasilj et al., 2012 (link)). Protein identification was performed with Mascot V2.2 (Matrixscience, London, United Kingdom), and determination of peptide ion signals for label-free quantification was performed with Progenesis LCMS V2.6 (Nonlinear Dynamics, Newcastle on Tyne, United Kingdom); relative quantification was based on the most intense three signals (MI3; Silva et al., 2006 (link); Groessl et al., 2012 (link)). Isoform specificity of peptides was obtained from the proteomicsdb database (www.proteomicsdb.org;Wilhelm et al., 2014 (link)).

Parotid glands were excised and protein was isolated as described above. A Direct IP Kit (Thermo Fisher Scientific) was then used per the manufacturer's specifications. Briefly, 1 mg of protein lysate was added to a spin column containing an Agarose Resin slurry (provided in IP Kit) and incubated at 4°C for one hour. Anti-Ambra1 (Cell Signaling) was then added and incubated at 4°C overnight. On the second day, the antibody/lysate solution was added to a spin column containing Protein A/G agarose (provided in IP Kit). The spin column and antibody/lysate were then washed with a Lysis Wash Buffer and Conditioning Buffer (provided in IP Kit). Next, a sample buffer elution was prepared using 5× Lane Reducing Buffer (provided in IP Kit), dithiothreitol (DTT), and deionized water. This sample buffer elution was added to the antibody/lysate solution and then loaded into a 12% SDS-PAGE gel and run overnight as described above. The membranes were blocked using 5% BSA and then immunoblotted with Ambra1, Beclin-1, and Bcl-2.