Cell Signaling Transduction Profiling

Cell lines profiled for signaling transduction analysis were seeded in technical replicates (n = 6) in 6-well plates and cultured until 80% confluent. Cells were washed twice with PBS (Invitrogen Life Technologies, Carlsbad, CA, USA) and lysed in Tissue Protein Extraction Reagent (T-PER) (Thermo Fisher Scientific, Waltham, MA, USA) supplemented with 300 mM sodium chloride and a mixture of protease and phosphatase inhibitors to prevent protein degradation and preserve the integrity of the phosphoproteome, as previously described [19 (link)]. Protein concentration in each sample was assessed using the Coomassie Protein Assay Kit (Thermo Fisher Scientific, Waltham, MA, USA) following manufacturer’s instructions. Lysates were first diluted to 1 µg/µL in T-PER and subsequently brought to a final concentration of 0.5 µg/µL in 2X Tris-Glycine SDS Sample buffer (Invitrogen Life Technologies, Carlsbad, CA, USA) supplemented with 5% β-mercaptoethanol (Sigma-Aldrich, St. Louis, MO, USA). Lastly, lysates were boiled for 8 min at 100 °C and stored at −80 °C.