Pan akt

The following antibodies were from Cell Signaling (Danvers, MA): pan-AKT(#4685), AKT1(#2938), pAKT Ser-473(#9271, #4060), PARP (#9542), GSK3α (#9338), pGSK-3α ser21 (#9316), GSK3β (#9315), pGSK3β ser9 (#9323), pS6 ribosomal protein ser240/244 (#5364) all used at 1:1000 dilution. Antibodies against α-tubulin (#05-829) and AKT3 (#07-383) were from Millipore (Billerica, MA) and were used at 1 μg/ml and 1:500, respectively.
The GBM cell lines, U87 and U251, were obtained from ATTC (Manassas, VA) and maintained in DMEM (Waymouth medium, Life Technologies; Grand Island, NY) supplemented with 10 % fetal calf serum (Life Technologies), at 37 °C, 5 % CO₂ in a humidified incubator. They were routinely monitored for mycoplasma infection using fluorescence microscopy after DAPI staining to detect extranuclear nucleic acids.

Cell lysis and western blotting was performed as previously described.^{14 (link), 41 (link)} Membranes were probed with the following primary antibodies: phospho Akt S473 (#9271), pan-Akt (#4691, Cell Signaling), β-Actin (#A1978, Sigma Aldrich), α-Tubulin (#05-829, Millipore, Billerica, MA, USA), PR (#SC-7208X, Santa Cruz, Santa Cruz, CA, USA), TBP (#ab818, Abcam), and 14-3-3σ (#A301-648A, Bethyl Laboratories, Montgomery, TX, USA). Membranes were developed using ECL Reagents (Life Technologies) and protein bands were visualized with a Fuji Film LAS-3000 Imaging System (Stamford, CT, USA).

The PIK3CA point mutations identified in ctDNA and FFPE tumor tissue were introduced into the full-length PIK3CA coding sequence using site-directed mutagenesis and were inserted into an expression vector (RC213112, Origene). Sequences of the primers used to construct PIK3CA mutation expression vectors are provided in Supplementary Materials and Methods. A human colorectal cancer cell line (DiFi) was transfected with the PIK3CA mutation expression vectors and assessed by Western blot to identify changes in the phosphorylation levels of AKT and other downstream EGFR targets. The following antibodies were used: Flag (Sigma-Aldrich, Cat. # F3165), p-AKT (Ser473) (Cell Signal, Cat. # 4060), pan-AKT (Cell Signal, Cat. # 4685), p-ERK1/2 (Cell Signal, Cat. # 4367), and total ERK1/2 (Cell Signal, Cat. # 9107). Cell viability analyses were performed to detect sensitivity to cetuximab and/or to 5-fluorouracil (5-FU). The hotspot mutations in PIK3CA exons 9 and 20 (p.E542K, p.E545K and p.H1047R) were included for comparison. Vector construction, western blot and cell viability analysis are described in the Supplementary Materials and Methods.