Complete protease

Isolated TIM3⁺ and TIM3^- NK cells were collected and lysed directly in RIPA buffer supplemented with complete protease (Roche, Basel, Switzerland) and phosphatase inhibitors (Solarbio Science & Technology, Beijing, China). Protein levels in the lysates were quantified using a BCA kit. Proteins were separated using 4%–20% Precast-Gel and transferred to nitrocellulose (NC) membranes (Pall Corporation, New York, NY, USA). The membranes were blocked with 10% skimmed milk (Chuntest Biotechnology, Shanghai, China) and subsequently incubated with anti-AKT, anti-Phospho-AKT, and anti-GAPDH antibodies (CST, Danvers, MA, USA) at a 1: 1000dilution. The antibodies were dissolved in a solution containing 5% dried milk in Tris-buffered saline with TBS-T (20 mmol/L Tris-HCl buffer, pH7.4, 150 mmol/L NaCl, 0.05% Tween 20). After extensive washing with PBS, the membranes were incubated with relevant horseradish peroxidase-conjugated secondary antibodies (1: 5000 dilution; CST). The labeled protein bands were detected using Super ECL Plus Detection Reagent. All protein levels were normalized to GAPDH.

Protein extracts were prepared using modified RIPA buffer (50 mM Tris HCL pH 7.4, 0.5% NP-40, 0.5% w/v Na-deoxycholate, 150 mM NaCl with complete protease (Roche, Welwyn Garden City, UK). N-ethylmaleimide (25 mM, Sigma) was also added to the RIPA buffer for immunoprecipitation of Keap1 to look at ubiquitin. Nuclear extraction was performed using Active Motif kit (Rixensart, Belgium). Immunoprecipitation was conducted with anti-Keap1 or anti-Nrf2 in 500-1000 µg of cell lysate overnight at 4°C. Immunoprecipitates were captured with mouse TrueBlot IP beads (eBioscience, Hatfield, UK). After extensive washing, bound proteins were released by boiling in SDS–PAGE sample buffer. Protein extracts (40 µg or immunoprecipitates) were analysed by SDS-PAGE (Invitrogen, Paisley, UK) and detected with Western blot analysis by chemiluminescence (ECL Plus; GE Healthcare, Hatfield, UK). Immunoprecipitation of BEAS2Bs treated with Biotin-Arachidonic acid was conducted with Neutravidin Agarose Resin in 750 µg of cell lysate overnight at 4°C.Protein expression levels were expressed relative to β-actin or TBP expression.

For testing shRNA knockdown efficiency targeting each clock gene, the cDNA was cloned into a p3XFlag-CMV-14 vector. Flag-tagged cDNA was co-transfected with the indicated shRNA in 3T3 or 293T cells. Forty eight hours post-transfection, cells were lysed in RIPA buffer containing complete protease (Roche) and phosphatase inhibitors (Sigma). Protein expression was determined by Western blot analysis using an anti-Flag monoclonal antibody (Sigma). For all Western assays, PVDF membrane was used in protein transfer, and SuperSignal West Pico substrate (Thermo Scientific) was used for chemiluminescent detection.

Cells were lysed (50 mM Tris, 150 mM NaCl, 1 mM EDTA, 1% Triton-X100, 1 mM DTT, cOmplete protease (Roche) and PhosSTOP phosphatase inhibitor (Roche)) after indicated treatment and run on Criterion XT Tris-HCl gel 4%–15% gradient (Bio-Rad). Following transfer to PVDF membrane and blocking in 5% non-fat dry milk in TBST, membranes were incubated with antibody overnight. After incubation with secondary antibody, chemiluminescence was used to visualize bands.

HEK293T cells were lysed 48 h post-transfection in lysis buffer (25 mM Tris–HCl, pH 8.0, 150 mM NaCl, 2% Nonidet P-40, supplemented with complete protease (Roche) and Phospho-STOP (Sigma) inhibitor mixtures) for 30 min on ice and cleared by centrifugation (10 min, 20,000×g). Protein concentration of the supernatant was determined using a bicinchoninic acid (BCA) protein assay (Pierce™). 1 mg/ml of all cell lines was incubated overnight in the presence of protein G Dynabeads™ (Thermo Fisher Scientific) and HA antibody (Cell signaling, 3724) or normal rabbit IgG antibody (Santa Cruz, sc-2027) at 4 °C on a rotating device. Afterwards, the beads were collected and washed repeatedly with the lysis buffer. Finally, beads were re-suspended in lysis buffer supplemented with 4 × LDS sample buffer and 100 mM DTT, boiled for 10 min at 95 °C, and loaded on 4–12% Bis–Tris NuPAGE gels (Life Technologies).