Updated PubMed. ABIN1 KO RAW264.7 cells and stably reconstituted with 3×FLAG-ABIN1 WT or D485N were subjected to mass spectrometric analysis. Three biological replicates were prepared for each condition. Cells were stimulated for 3 hours with 100 ng/ml LPS. ABIN1 was immunoprecipitated with 30 uL anti-FLAG M2 Magnetic Beads (M8823, Sigma-Aldrich) for 2 h at 4℃. Immunoprecipitated sample was eluted by incubation at 90°C for 5 min in 100 uL PTS buffer. Each sample was split into 3 tubes for protein digestion with Lys-C and trypsin, chymotrypsin, or Glu-C.