Horseradish peroxidase linked secondary anti rabbit or anti mouse antibodies

Cells were lysed in an ice-cold RIPA lysis buffer containing a cocktail of proteinase inhibitors and phosphatase inhibitors. The protein concentration in the supernatant was determined using the BCA protein assay (Pierce Chemical). Samples were subjected to 8% to 15% SDS-PAGE, and the separated proteins were electrophoretically transferred to PVDF membranes. Blots were incubated with the primary antibodies, and then incubated with horseradish peroxidase-linked secondary anti-rabbit or anti-mouse antibodies (GE). GSC protein lysate samples were probed with 279 validated primary antibodies for the analysis at the MD Anderson Functional Proteomics Reverse Phase Protein Array (RPPA) Core facility. (https://www.mdanderson.org/education-and-research/resources-for-professionals/scientific-resources/core-facilities-and-services/functional-proteomics-rppa-core/index.html).

The cell lysate was prepared using Complete Lysis-M (Roche, Basel, Switzerland) solution containing a cocktail of proteinase inhibitors and phosphatase inhibitors. The protein concentration was determined using the BCA protein assay (Pierce Chemical, Dallas, TX, USA). Samples were subjected to 4% to 20% Mini-PROTEAN TGX (BioRad, Hercules CA, USA) PAGE and transferred to PVDF membranes using Trans-Blot Turbo Transfer System (BioRad). The blots were probed with primary antibodies [STAT1 (CST, Cat: CST-14994, Danvers, MA, USA), phosphoSTAT1 (CST, Cat: CST-9167), Akt (CST, Cat: CST-9272), phosphoAkt (CST, Cat: CST-4060), p38α MAPK (CST, Cat: CST-9228), phospho-p38α MAPK (CST, Cat: CST-4511), phospho-mTOR (CST, Cat: CST-5536) and β-Actin (CST, Cat: CST-3700)] and then incubated with horseradish peroxidase-linked secondary anti-rabbit or anti-mouse antibodies (GE, Piscataway, NJ, USA). Original figures of western blot have been included as Supplementary Figure S5 and antibodies dilutions have been provided as Supplementary Table S1.