Pierce bicinchoninic acid bca kit

Determination of ∆6-desaturase (∆6-D) protein levels was performed in liver tissue samples from rainbow trout using a Fish Fatty Acid Desaturase 2 ELISA Kit (MBS066226, MyBiosource Inc., San Diego, CA, USA; purchased from Biozol, Eching, Germany) according to the manufacturer’s instructions. In brief, tissue samples were weighted on dry ice and lysed in phosphate buffered saline using a TissueLyser II (Qiagen, Hilden, Germany). Following centrifugation, supernatants were applied to the Microelisa multiwall plate provided by the ∆6-D Kit. Samples were incubated with a HRP (horseradish peroxidase)-conjugate reagent followed by several washing steps. Protein concentration was quantified following HRP-mediated color reaction (consecutive application of Chromogen A, B and Stop solutions) by absorbance measurements at 450 nm using a Labsystems iEMS MF multiplate reader (MTX Lab Systems, Bradenton, FL, USA purchased from Thermo Fisher Scientific, Darmstadt, Germany). ∆6-D protein concentration was calculated using a standard curve. ∆6-D concentrations were normalized to total protein concentrations, which were evaluated via the Pierce bicinchoninic acid (BCA) kit (Thermo Fisher Scientific) according to the manufacturer’s protocol, respectively.

All fibroblast cell lines were tested for Mycoplasma. Skin fibroblasts were cultured at 37°C in 5% CO₂ in Dulbecco's Modified Eagle Medium (DMEM) 3.7 g/L NaHCO₃ (HyClone) supplemented with 10% Fetal Bovine Serum (FBS) and 1% penicillin/streptomycin. Protein was extracted from cultured fibroblasts by resuspending in radioimmunoprecipitation assay (RIPA) buffer with protease inhibitor cocktail (Roche), then sonicated and incubated on ice for 30 min before centrifugation at 18000g for 20 min at 4°C. Total protein concentration was determined with the Pierce Bicinchoninic acid (BCA) kit (ThermoFisher) following the manufacturer's protocol. Protein lysates containing 20 μg of protein samples were analyzed by SDS‐PAGE Western blot (BioRad system) using primary antibodies against NBAS (1:500, ThermoFisher, PA5‐103963), USE1 (p31) (1:250 Sigma Aldrich, HPA026851) and GAPDH (1:10 000; Sigma Aldrich, G9545), and detected by anti‐rabbit IgG secondary‐horseradish peroxidase (HRP) conjugated antibody (1:5000, Cell Signaling Technology, #7074S) using Enhanced chemiluminescence reagents (ECL) (GE Healthcare). Protein band intensities were quantified using ImageJ software and normalized to GAPDH.

We performed western blot assays to determine expression of PD-1 and CXCR4 in PDAC cells and PDOs. The cells were lysed with RIPA lysis buffer (Cell Signaling Technology) supplemented with phosphatase and protease inhibitors (Sigma-Aldrich). Protein concentrations were determined using the Pierce^™ bicinchoninic acid (BCA) kit (ThermoFisher). Protein samples (20–40 μg) were electrophoresed in 10% SDS polyacrylamide gels and transferred to polyvinylidene difluoride membranes (Bio-Rad). The membranes were blocked with 5% milk and incubated overnight at 4°C with primary antibodies against PD-1 (1:1000, Proteintech, 66220-1-Ig), CXCR4 (1:1000, Proteintech, 60042-1-Ig), or β-actin (1:5000, Sigma-Aldrich) for loading control. Then the blots were incubated for 1 h at room temperature with a corresponding HRP-conjugated secondary antibody (1:5000; Santa Cruz Biotechnology), visualized in ECL solution (SuperSignal West Pico Chemiluminescent Substrate, ThermoFisher) and exposed with an UVP ChemiDoc-It2imager. Blots were quantified using ImageJ (NIH) and results analyzed using GraphPad software.

Radio immunoprecipitation assay lysis buffer was used to homogenize the LHb sample on ice, and then the samples were centrifuged at 12,000 × rpm for 20 min. The Pierce bicinchoninic acid (BCA) kit (Thermo Scientific, Rockford, IL) was used to measure the protein concentration in the supernatant. The samples were boiled with protein loading buffer, and 30 μg total protein was separated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and then transferred onto polyvinylidene fluoride (PVDF) membranes. The PVDF membranes were blocked with 5% skim milk in TBST (20 mM Tris–HCl, pH 7.5, 150 mM NaCl, and 0.05% Tween-20) for 2 h at room temperature. Membranes were incubated with primary antibodies against p-CaMKII (Thr286, 1:1000 dilution, SC-12886-R, Santa Cruz Biotechnology, Dallas, TX, USA) and GAPDH (1:10,000 dilution, 51,332, Proteintech, Manchester, UK) at 4 °C overnight. The PVDF membranes were washed using TBST and incubated with the HRP-conjugated goat anti-rabbit IgG (H + L) secondary antibody (1:10,000 dilution, SA00001–2, Proteintech) for 1 h at room temperature. An ImageQuant LAS4000 mini image analyzer (GE Healthcare, UK) was used to capture the images, and the Quantity One Analysis Software (Version 4.6.2, Bio-Rad Laboratories, Hercules, USA) was used to quantify the band intensities.