Using L3MBTL3 knockout and rescued stable human breast cancer cell line (MDA-MB-231) to detect the novel interacting proteins of L3MBTL3 by immunoaffinity purification and mass spectrometry. Identification of L3MBTL3 interaction proteins in the breast cancer cell.

We used L3MBTL3 knockout MDA-MB-231 cell (L3-KO5+V) and L3MBTL3 rescue MDA-MB-231 cell (L3-KO5+L3). The cells were collected by centrifugation and lysed in lysis buffer (50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 1 mM EDTA, 0.1% NP40, 8% glycerol, protease inhibitor cocktail (#P1010, Beyotime, China) on ice for 30 min. Then the cell lysate supernatant was incubated with M2-conjugate agarose beads (#A2220, Merck, USA) by rotation overnight at 4°C. After washing, the beads were eluted by 3×FLAG peptide (#F4799, Merck, USA). Then the elution protein was verified using silver staining, then the target lane was excised and subjected to analyze by an EASY-nLC 1200 UHPLC system (ThermoFisher Scientific, USA) coupled to a Q Exactive HF-X mass spectrometer (ThermoFisher Scientific, USA).