TAS1440, an LSD1 inhibitor, activates TGF-β and NOTCH signaling in neuroendocrine small-cell lung cancer

NCI-H1417 cells were electrotransfected with either a pcDNA3-3×Flag-myc-empty or pcDNA3-3×Flag-myc-LSD1. Cells were treated with different LSD1 inhibitors for 2 h, harvested. Lysates were immunoprecipitation by using ANTI-Flag® M2 Affinity Agarose Gel (Cat. No.‎ A2220, Sigma-Aldrich) overnight at 4°C. After washing and eluting by 3× FLAG® peptide (Cat. No.‎ F4799, Sigma-Aldrich). Eluted samples were precipitated using acetone. The pellet was dissolved in 1× SDS sample buffer and resolved by SDS–PAGE. The Bands were excised from the gel, and trypsin-digested samples were subjected to liquid chromatography-tandem mass spectrometry (LC-MS/MS) using an Orbitrap Velos Pro (Thermo Fisher Scientific). The resulting LC-MS/MS dataset was analyzed using Mascot software (version 2.5.1, Matrix Science, Boston, MA, USA). The Mascot score indicates the probability that the observed match was a random event (a Mascot score > 100 indicates that the absolute probability is <1 × 10−10).