Proteinase and phosphatase inhibitor

After normoxic or hypoxic incubation, cells were collected by using a cell-collecting scraper. Collected samples were washed twice with ice-cold PBS prior to incubation in RIPA lysis buffer (Thermo Fisher) containing proteinase and phosphatase inhibitor (Thermo Fisher) for 30 min on ice. Next, the lysates were centrifugated for clearance (15 000 r.p.m. at 4 °C for 30 min). The protein concentration in the lysate was determined by using a bicinchoninic acid assay kit (Bio-Rad, Hercules, CA, USA). Obtained protein (10 μg) was then loaded in 10% SDS-polyacryl-amide gel for electrophoresis and transferred to a polyvinylidene fluoride (PVDF) membrane. The protein-transferred membrane was washed with tris-buffered saline containing a 0.1% Tween-20 (TBST) solution (10 mM Tris-HCl (pH 7.6), 150 mM NaCl, and 0.1% Tween-20) and blocked with 5% skim milk or 5% bovine serum albumin for 15 min. The membranes were then washed with TBST solution for 30 min and incubated with primary antibody (1:1000 dilution) overnight at 4 °C. The membranes were washed again and then incubated with HRP-conjugated secondary antibody (1:10 000 dilution) for 6 h at 4 °C. The western blotting bands were detected by using enhanced chemiluminescence (Bio-Rad). Densitometric analysis of western blotting bands was quantified by using ImageJ software.

Primary mouse skin fibroblasts were lysed by RIPA solution and fresh tissue was lysed using T-PER reagent with proteinase and phosphatase inhibitor (Thermo Fisher). After gel electrophoresis and electrotransferation, proteins were detected with antibodies against SHIP-1, CK1α, pAkt, Akt (pan), β-catenin, pErk, pJNK, pSmad2/3, pp38 and GAPDH. HRP conjugated goat anti-rabbit or anti-rat secondary antibodies were used. Semiquantitative analysis based on densitometry was performed using Image J software (National Institute of Health, Bethesda, MD, USA).

EGFRvIII-CT-2A tumor-bearing C57BL/6 mice received lymphodepletion followed by adoptive transfer of either WT or IFN-γ^−/− CAR T-cells plus a single dose of vaccination. Mice were euthanized and tumors isolated at day 7 post vaccination. Tumors were weighted, cut using a razor blade into small pieces and dounced to generate tumor homogenate in tissue protein extraction buffer (T-PERTM, Thermo Fisher Scientific, cat. no. 78510) in the presence of 1% proteinase and phosphatase inhibitors (Thermo Fisher Scientific, cat. no. 78442). The lysates were incubated at 4°C for 30 min with slow rotation followed by top-speed centrifugation to remove debris. The supernatants were transferred to a clean tube and stored at −80°C. Part of the samples were subjected to Luminex analysis using a Mouse Cytokine 32-Plex panel analysis at Eve Technology.