Coimmunoprecipitation was performed by lysing cells in tris buffer [10 mM tris (pH 8.0) (Santa Cruz Biotechnology, sc-3715A), 150 mM NaCl (Sigma-Aldrich, S7653), 10% glycerol (Sigma-Aldrich, G7757), 0.5% NP-40 (Sigma-Aldrich, 56741)] or in CHAPS buffer, in the case of ULK1, as previously described (31). Radioimmunoprecipitation assay (RIPA) buffer [150 mM NaCl (Sigma-Aldrich, S7653), 1% NP-40 (Sigma-Aldrich, 56741), 0.5% deoxycholic acid (MP Biomedicals, 101496), 0.1% SDS (Sigma-Aldrich, L3771), 50 mM tris (pH 8.0) (Santa Cruz Biotechnology, sc-3715A), and 2 mM MgCl2 (Sigma-Aldrich, M8266)] was used to analyze ubiquitination levels of immunoprecipitated proteins and for immunoblotting assays. Lysis buffer was complemented with protease and phosphatase inhibitors [Protease Inhibitor Cocktail plus (Sigma-Aldrich, P8340), 5 mM sodium fluoride (Sigma-Aldrich, S-7920), 0.5 mM sodium orthovanadate (Sigma-Aldrich, S6508), 1 mM sodium molibdate (Sigma-Aldrich, S-6646), 50 mM 2-chloroacetamide (Sigma-Aldrich, C0267), 2 mM 1,10-phenanthroline monohydrate (Sigma-Aldrich, 320056), and 0.5 mM phenylmethylsulfonyl fluoride (Sigma-Aldrich, P7626)]. Coimmunoprecipitation was performed with 1 mg of protein extracts from transfected cells or 3 mg in the case of endogenous proteins assays. For endogenous protein immunoprecipitation, protein extracts were incubated overnight with 2 μg of antibody, and immunocomplexes were recovered using 25 μl of Protein A Sepharose (GE Healthcare, GE 17-1279-01). For immunoprecipitation of overexpressed tagged proteins, protein extracts were incubated with 25 μl of anti-FLAG, anti-HA, or anti-MYC antibodies conjugated to agarose beads (Sigma-Aldrich: A22220, A2095, and A7470, respectively) for 2 hours. To test the covalent binding of polyubiquitin chains to ULK1, in the first-round immunoprecipitation assay, cell extracts were prepared as described above, and then, lysates were incubated with the anti-FLAG antibody conjugated to agarose beads for 2 hours. Immunoprecipitates were washed three times with RIPA buffer. Before the second-round immunoprecipitation assay, the immunoprecipitates were denatured by boiling for 5 min at 95°C in the lysis buffer containing 1% SDS. The eluates were then diluted 1:10 with lysis buffer and reimmunoprecipitated with the anti-FLAG antibody conjugated to agarose beads for 2 hours.

Immunocomplexes were separated on NuPAGE bis-tris gels (Life Technologies, 4 to 12% EA0378BOX and 3 to 8% NW04120BOX) and electroblotted onto nitrocellulose (Whatman Amersham, 10600041) or polyvinylidene difluoride (Millipore, IPVH20200) membranes. Detection was achieved using horseradish peroxidase–conjugated secondary antibodies [anti-goat 705-036-147, anti-rabbit 711-036-152, and anti-mouse 715-036-150 (Jackson ImmunoResearch Laboratories)] and enhanced chemiluminesence (ECL) [Immobilon Classico WBLUC0500 and Immobilon Crescendo Western HRP substrate WBLUR0500 (Millipore)]. Signals were acquired using Amersham Hyperfilm ECL (GE Healthcare, 28-9068-37) or a ChemiDoc imaging system.

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