Column oven

Peptides were incubated in RKO cells (ATCC, #CRL-2577) as follows: Experiment 1: RKO cells were incubated in 25 µM compound for 90 min and 24 h. Experiment 2: RKO cells were incubated in 25 µM for 30 min or 72 h. Following peptide incubation, the cells were washed twice with PBS, trypsinized and pelleted. The pellets were lysed in RIPA buffer (Perkin Elmer) for total lysis, or CelLytic NuCLEAR Extraction Kit (SIGMA) for subcellular fractionation. Total protein quantification BCA (Pierce) was performed prior to MS analysis to normalize sample data. Total and fractionated cell lysates were analysed by quantitative UPLC-MS utilizing a Waters Xevo TQ-XS (WBA0259) and an Acquity UPLC system from Waters consisting of Sample Manager (M16UFL953M), Acquity PDA (F17UPD457A), Column Oven (E17CMP703G) and Binary Solvent Manager (E17BUR621G). The Waters TQ-XS was operated in +ve ion Electrospray (ESI) mode with the optimized transitions for peptides 4, 4E, 7, and TAT. The chromatograms at each transition were extracted, smoothed, and integrated to give the standard curve. Chromatograms of the samples were treated similarly, and by linear regression (1/x) an in-cell concentration was established in the re-suspended cell lysate using the Waters MassLynx TargetLynx^TM product.

NCI-H460 and LK2 cells were treated with 10 μM AZD4785. Cells were trypsinized, washed three times with ice-cold PBS and counted, before lysing in RIPA buffer as described previously. For cell fractionation experiments, cells were processed using the ThermoFisher subcellular fractionation kit (78840). Protein was quantified using BCA assay (Pierce). Samples were analysed by UPLC-MS utilizing a Waters Xevo TQ-XS (WBA0259) and an Acquity UPLC system from Waters consisting of Sample Manager (M16UFL953M), Acquity PDA (F17UPD457A), Column Oven (E17CMP703G) and Binary Solvent Manager (E17BUR621G). Full mass spectrometry optimization and methods can be found in Supplementary Figure S1.