Quantification of Phosphoprotein Signaling

Cells were cultured in complete media unless otherwise noted. For measuring relative changes in phospho-AKT, phospho-S6K, and phospho-ERK level in response to inhibitors, HUVECs were cultured in media with inhibitor for 24 hours. Cells were lysed on ice with radioimmunoprecipitation assay buffer (Thermo Fisher Scientific) containing Halt protease and phosphatase inhibitor (Thermo Fisher Scientific). Clarified lysates were resolved on Novex 4 to 12% bis-tris gel (Thermo Fisher Scientific) and transferred to a polyvinylidene difluoride membrane. Standard immunoblotting protocols were performed for Western blotting with primary antibodies used at the following concentrations: anti-p110α (1:1000; Cell Signaling Technology, #4249), anti–p-AKT-Thr³⁰⁸ (1:1000, Cell Signaling Technology, #9275), anti–p-AKT-Ser⁴⁷³ (1:1000; Cell Signaling Technology, #9271), anti-AKT (1:1000; Cell Signaling Technology, #9272), anti–pS6K-Thr³⁸⁹ (1:1000; Cell Signaling Technology, #9205), anti-S6K (1:1000; Cell Signaling Technology, #9202), anti–pERK1/2-Thr²⁰²/Tyr²⁰⁴ (1:1000; Cell Signaling Technology, #9101), anti-ERK1/2 (1:1000; Cell Signaling Technology), anti-Rac1/2/3 (1:1000; Cell Signaling Technology, #2465), and anti–glyceraldehyde-3-phosphate dehydrogenase (1:2000; Cell Signaling Technology, #2118). Horseradish peroxidase–conjugated secondary antibodies and SuperSignal West Femto or Clarity Western ECL chemiluminescent substrate were used for detection. Western blot images were quantified with Fiji/ImageJ. Proteome profiler human protease array (R&D Systems, #ARY021B) was performed according to the manufacturer’s instructions. Ras, Rac, and Cdc42 pulldown assays (Cytoskeleton Inc.) were performed according to the provided instruction manual. Average pixel intensity of each analyte and background signal were quantified using Fiji/ImageJ. Average pixel intensity was subtracted with average background signal. Expression level of each analyte was normalized by setting the lowest average pixel intensity and the highest average pixel intensity of each dataset to 0 and 100, respectively. Lysates from confluent cells cultured in complete media were used to compare Rac–guanosine 5′-triphosphate (GTP) level in GFP control and PIK3CA mutant cells. For alpelisib treatment, confluent cells cultured overnight in reduced serum conditions were stimulated with complete media with or without alpelisib for 10 min. Cells were rinsed once with cold PBS++ and lysed with cold lysis buffer [50 mM tris (pH 7.5), 10 mM MgCl₂, 0.3 M NaCl, 2% IGEPAL, and protease and phosphatase inhibitor cocktails, Cytoskeleton Inc.]. Lysates were sonicated at 3 W on ice for 10 s and clarified at 14,000g for 5 min. Protein concentration was quantified with bicinchoninic acid assay (Thermo Fisher Scientific), and the protein concentration and volume were equalized with cell lysis buffer. Five hundred micrograms of lysate was incubated with 10 μg of PAK-p21 bidning domain (PAK-PBD) or RAF-Ras binding domain (RAF-RBD) beads on a rotator for an hour at 4°C. Bead pellets were washed three times with wash buffer [25 mM tris (pH 7.5), 30 mM MgCl₂, and 40 mM NaCl, Cytoskeleton Inc.] and extracted with 2× NuPAGE lithium dodecyl sulfate (LDS) containing 100 mM dithiothreitol. For quantification, active RAC and RAS1 were normalized to total RAC and RAS1, respectively.