LC-MS/MS spectrometry analysis of PLCE1.

Protein pulldown was performed using a previously reported method (17). Briefly, HEK293 cells were transfected with a plasmid expressing PLCE1-Myc or TAK1. 24 h post-transfection, cells were harvested for preparing total cell lysates, which was then subjected to protein pulldown assay using the Myc-Tag beads (Sepharose® Bead Conjugate; #55464; CST, Beverley, MA, USA). After 4 h-incubation on a rotator, unbound proteins were removed using the ice-cold wash buffer. Finally, the resulting immunocomplex was subjected to LC-MS/MS spectrometry analysis (Shanghai Applied Protein Technology Co., Ltd.). MS/MS spectra were searched using MASCOT engine (Matrix Science, London, UK; version 2.4) against the UniProKB human (161584 total entries, downloaded 20180105).