Epididymal fat tissues (200 mg each) derived from Gal-9wt/wt C57BL/6JJcl mice were minced, lysed in 500 μl of RIPA buffer (Thermo Scientific) containing 1 mM DTT under reducing condition, and immunoprecipitated with Protein G Immunoprecipitation kit (Sigma) by using anti-galectin-9 (Biolegend, Cat#137,902) or Purified rat IgG2a, κ isotype control (Biolegend, Cat#400,502) antibodies. The immunoprecipitated protein complexes were electrophoresed in SuperSep Ace 15–20% Tricine Gel (GE Healthcare), and gel-digested with trypsin. The samples were lyophilized, suspended in 20 μl of 0.1% formic acid, and subjected to nanoLC-MS/MS using UltiMate 3000 HPLC, Q-Exactive Plus, and Xcalibur (Thermo Scientific). The proteins were searched and identified by Mascot (Matrix Science, Boston, MA). The scores by Mowse scoring algorithm, hit peptide fragment numbers, the exponentially modified protein abundance index (emPAI) were analysed. The gene annotation was performed by PANTHER 15.0 (http://www.pantherdb.org/ ).
The lysates from epididymal fat tissues of Gal-9wt/wt and Gal-9−/− C57BL/6JJcl mice were also immunoprecipitated with anti-peroxiredoxin 2 (PRDX2) antibody (abcam, Cat#ab109367), and subjected with Western blot analysis with anti-galectin-9 antibody (Biolegend, Cat#137,902).
The lysates from epididymal fat tissues of Gal-9wt/wt and Gal-9−/− C57BL/6JJcl mice were also immunoprecipitated with anti-peroxiredoxin 2 (PRDX2) antibody (abcam, Cat#ab109367), and subjected with Western blot analysis with anti-galectin-9 antibody (Biolegend, Cat#137,902).