Pi3 kinase p110α

To ensure equal sample loading, protein concentration was determined using bicinchoninic acid (BCA) protein assay reagent (Sigma, UK) and adjusted accordingly. NuPAGE LDS Sample Buffer (4X) (Thermofisher Scientific, UK) plus 5% β-mercaptoethanol were added to the samples, which were subsequently denatured by heating to 100 °C for 10 min. Samples were then loaded on NuPAGE Novex 10% Bis–Tris protein gels (Thermofisher scientific, UK) using the Mini Protean III system (Bio-Rad, UK). Proteins were transferred onto nitrocellulose blotting membrane (GE Healthcare Life Sciences, UK) through wet transfer in a Bio-Rad Mini Trans-Blot. The membranes were blocked for 1 h using 5% bovine serum albumin/PBS tween and subsequently incubated with appropriate primary antibodies at 4 °C overnight.
The following primary antibodies being used were acquired from Cell Signalling Technology: Akt (#9272), Phospho-Akt (Ser473) (#9271), Phospho-Akt (Thr308) (#2965), and PI3 Kinase p110α (#4249). Anti-GAPDH (mAbcam, #9484) was used as loading control. After overnight incubation, membranes were probed with secondary antibodies. Levels of protein were finally quantified using the Odyssey imaging system from Li-Cor Biosciences (Image Studio Lite Ver 5.2).

Cell and tissues lysates were collected as previously described (29 (link)). Protein concentrations were determined using BCA Assay (Beyotime Biotechnology). Equal amounts of protein were separated with 8–12% SDS-PAGE and then electrophoretically transferred onto a polyvinylidene difluoride membrane (Millipore, Billerica, MA, USA). TBST containing with 5% non-fat milk or bovine serum albumin was used to block non-specific binding for 2 h at room temperature. Then, membranes were incubated with primary antibodies according to the instructions overnight at 4°C followed by appropriate secondary antibodies. Signals generated by enhanced chemiluminescence (Millipore) were recorded with a CCD camera (Tanon, Shanghai). The experiments were performed three biological repeats. Primary and secondary antibodies included: ALDH1A3 (1:1,000, Sigma-Aldrich, #HPA046271), HK2 (1:1,000, Cell Signaling Technology, #2867), PKM2 (1:1,000, Cell Signaling Technology, #4053), PPARγ (1:1,000, Santa Cruze, #sc-7273), mTOR (1:1,000, Cell Signaling Technology, #2983), p-mTOR (1:1,000, Cell Signaling Technology, #5536), PI3 Kinase p110α (1:1,000, Cell Signaling Technology, #4249), Akt (1:1,000, Cell Signaling Technology, #2920), p-Akt (1:1,000, Cell Signaling Technology, #4060), β-actin (1:5,000, Sigma-Aldrich, #5441).

PLC/PRF/5 and HepG2 cell lines were cultured in presence of regorafenib, sorafenib, GSK1838705A and ramucirumab alone or in combination for 48h. According to the western blotting protocol above described, the levels of the protein investigated were evaluated with the following antibodies: Phospho-PI3K p85(Tyr458)/p55(Tyr199) and PI3Kinase p110α, Phospho-Akt (Ser473) and Akt (pan) (11E7), β-Actin (13E5) (Cell Signaling). Muse PI3K Activation Dual Detection Kit (Millipore), was used to observe the fluorescent signal emitted by a specific anti-phospho-Akt (Ser473), Alexa Fluor 555, and an Akt-PECy5 conjugated antibody. This two-color kit is useful to measure the extent of Akt phosphorylation relative to the total Akt expression. The levels of both the total and phosphorylated protein can be evaluated in the cell in the same time, resulting in a normalized measurement of Akt activation after treatments. PLC/PRF/5 and HepG2 cells were treated in presence of the indicated concentrations of regorafenib, sorafenib, GSK1838705A and ramucirumab alone or in combination for 24 h respecting the drug concentrations indicated above. Results were representative of three independent experiments.