Membrane protein extraction for LC-MS/MS analysis

A549, H1975, and CL1-5 cells (4 ×10^7) were treated with GMI (0.6 μM) for 24 h. Cells were washed with 1×cold PBS and harvested by scraper. Membrane proteins were isolated by using the Trident Membrane Protein Extraction kit (GeneTex, Hsinchu, Taiwan) according to the manufacturers instructions. The extract lysates were re-dissolved with an appropriate amount of 1×lysis buffer as membrane proteins. After that, the membrane proteins were lysed and reduced the disulfide bonds using TCEP (10 mM) and alkylated with CAA (40 mM). The samples were digested and prepared for LC-MS/MS as following previous study (Tseng AJ, Tu TH, Hua WJ, et al. Int J Biol Macromol 219: 940-948, 2022).