Silverquest

Affinity purification was performed using the AminoLink Plus Immobilization Kit (Pierce, Rockford, IL, USA) according to the manufacturer’s instructions. Briefly, NHS was diluted (1:10) in PBS (pH 7.4) containing complete proteinase inhibitor cocktail (Roche Biochemicals, Mannheim, Germany) and applied to the SE36-immobilized and non-SE36-immobilized (control) columns. The columns were subjected to end-over-end rotation for 1 h at room temperature. The columns were extensively washed with PBS containing the proteinase inhibitor cocktail followed by elution of bound proteins with 2 mL 0.1 M glycine-HCl buffer (pH 2.7). The binding proteins were eluted four times. Elutes were neutralized by 50 μL 1M Tris-HCl (pH 9.0) and subjected to sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and the bands were visualized by silver staining using SilverQuest (Thermo Fisher Scientific, Waltham, MA, USA) (Supplementary Fig. 2).

The co-immunoprecipitated protein complexes that were pulled down by His antibody or IgG control were separated on a 5–20% gradient SDS-PAGE gel, which was subsequently stained with Silver Quest (Thermo Fisher Scientific, USA). The identified protein bands were excised from the gel and subjected to in-gel digestion, in which the dried gel pieces were rehydrated and digested overnight at 30°C in 25 mM NH₄HCO₃ solution containing 12.5 ng/mL trypsin (Promega, USA). Peptide sequence analysis was performed on an Agilent LC system coupled to a Finnigan LTQ Mass spectrometry (Thermo Fisher Scientific, USA) at Core Facility of Molecular Biology, Institute of Biochemistry and Cell Biology (SIBCB).

SDS-PAGE-resolved, 4%–12% gradient gels were silver-stained with SilverQuest (Life Technologies), and appropriate bands were cut and subjected to in-gel digestion followed by liquid chromatography-MS with the Q Exactive HF mass spectrometer (Thermo Fisher Scientific). Resulting peptides were identified by protein-sequence database searches using MaxQuant software (Tyanova et al. 2016 (link)).

Purification of PPARγ protein was performed as previously described [16 (link)]. Briefly, we cross-linked 2 μg of antibody with 30 μL of Protein G Dynabeads (Invitrogen) in freshly dissolved 20 mM dimethyl pimelimidate (DMP) in a 0.2 M triethanolamine buffer (pH 8.2). The cross-linking reaction was performed for 1 h at room temperature. The reaction was stopped by replacing the buffer with 50 mM Tris-HCl (pH 7.5).
Cell lysates of 293T transfected with FLAG-PPARγ2 and Caco-2 cell (from a 10 cm dish and 48 dishes, resp.) were prepared with 1% NP-40 buffer (10 mM Tris [pH 7.8], 1 mM EDTA, 150 mM NaCl, and 1% NP-40). Lysates were subjected to immunoprecipitation with Protein G Dynabeads coupled to each antibody for 3 h. The immunoprecipitates were washed by 1% NP-40 buffer and eluted with 0.1 M glycine-HCl buffer (pH 2). The eluates were boiled with Laemmli sample buffer and then subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and then Colloidal Blue (Life Technologies), silver staining (Silver Quest, Life Technologies), or Western blotting with the indicated antibodies.