Alliance q9 advanced

Total protein was extracted by using RIPA lysis buffer. The equal amount of protein samples was analyzed on 10% SDS-PAGE by mini-Protein III system (Bio-Rad, USA). The proteins were transferred to polyvinylidene fluoride membrane (GE Healthcare, USA). The membrane was probed with mouse anti-human CD279 antibody (BioRad), rabbit anti-human pAKT (Thr308) (Cell Signaling Technology), rabbit anti-human AKT (Cell Signaling Technology) and rabbit anti-human GAPDH antibody (BioRad). The signals were detected in different exposure times depending on each protein by horseradish peroxide (HRP) conjugated secondary antibodies and enhanced chemiluminescence substrate with Alliance Q9 Advanced (UVITEC, UK) or X-ray film. All original unmanipulated Western blot images were provided as the Supplementary Information.

Cells were lysed in cold RIPA buffer (ab156034, Abcam) supplemented with protease and phosphatase inhibitors for 30 minutes in ice, and then centrifuged at 14,000 rpm for 20 minutes at 4°C. Supernatant was collected and protein quantification was performed using the Pierce BCA Protein Assay Kit (Thermo Fisher Scientific). Equal amounts of protein lysates were loaded onto a 10% polyacrylamide gel, transferred onto nitrocellulose membranes, and incubated for 1 hour in 5% nonfat dry milk at room temperature. Primary antibodies are reported in Supplementary Table S3. Blotted membranes were developed by using Immobilon Western Chemiluminescent horseradish peroxidase (HRP) Substrate (Merck Millipore) and imaged with UVITEC Alliance Q9 Advanced (UVITEC Cambridge).

Electrophoretic mobility shift assay (EMSA) for NFκB was performed with the SYBR Green & SYPRO Ruby EMSA stain kit (E33075) according to the manufacturer's instructions. (catalog no. E33075, Thermo Fisher Scientific). Briefly, cells were lysed in ice-cold cytoplasmic Buffer A added with protease and phosphatase inhibitors for 10 minutes. Lysates were centrifugated at 2,000 × g for 5 minutes, supernatant discarded, and nuclear pellet was then lysed in 70 μL buffer C added with protease and phosphatase inhibitors for 30 minutes. For oligomer duplexes, 500 nmol/L of each of the two complementary oligonucleotides was mixed in a total volume of 100 μL of binding buffer. Complementary oligonucleotides were NFκB F: 5′- AGTTGAGGGGACTTTCCCAGGC-3′ and NFκB R: 5′-GCCTGGGAAAGTCCCCTCAACT-3′. After 10 minutes incubation at 95°C followed by additional 15 minutes at 70°C, samples were left to form oligomer duplexes while gradually cooling to room temperature. A total of 20 μg of nuclear extract were then added in binding buffer for additional 30 minutes at room temperature. Protein–DNA complexes were resolved by native 4% PAGE. Nucleic acid staining was achieved by incubating gel with SYBR Green EMSA gel stain solution in shaking for 20 minutes. Nucleic acids were visualized using UVITEC Alliance Q9 Advanced (UVITEC Cambridge).