Nuclear and cytoplasmic protein extraction kit

Protein extraction was performed using the Nuclear and Cytoplasmic Protein Extraction kit (Thermo Scientific, Pierce Biotechnology, IL, USA) according to the manufacturer’s instructions. The samples were separated using 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and then transferred onto PVDF membranes (Bio-Rad, Hercules, CA, USA). Subsequently, the membrane was blocked in TBST (20 mM Tris base pH 7.6, 150 mM NaCl, 0.1% Tween-20) containing 5% non-fat milk for 4 h at room temperature. Then, the membrane was incubated with NF-kB (Invitrogen, Cat number MA5-15160) and Lamin B1 (Invitrogen, Cat number 33-2000) primary antibodies that were added to the blocking solution at 3 µg/mL, and the membrane was incubated at 4 °C overnight. After overnight incubation, the membrane was washed and then incubated with goat anti-rabbit immunoglobulin G HRP conjugated secondary antibodies (Invitrogen, Cat number G-21234) and goat anti-mouse immunoglobulin G HRP conjugated secondary antibodies (Invitrogen, Cat number PA1-86015) for an additional hour at room temperature. After washing three times with TBST (30 min each wash), proteins were detected by chemiluminescence and protein bands were analyzed by densitometry using ImageLab software (Bio-Rad 6.1, Hercules, CA, USA).

Zebrafish embryos were collected at the indicated stage and directly lysed in SDS sample buffer. For nuclear FOXO1 detection, cytoplasmic and nuclear protein samples were obtained from 48 hpf zebrafish embryos using Nuclear and Cytoplasmic Protein Extraction Kit (Thermo fisher) according to the manufacturer’s instruction. Tumor tissue lysates were prepared in RIPA buffer for 30 min on ice. HUVEC samples were directly harvested into SDS sample buffer. Proteins were separated by SDS-PAGE and blotted onto polyvinylidene fluoride membranes. The following antibodies were used: anti-MYC (Genomics Technology, SG4110-18, 1:1000), anti-a-Tubulin (Sigma, T9026, 1:1000), anti-GAPDH (Absci, 21612-2, 1:2000), anti-HIF1a (Ruiying Biological, RLT2133, 1:500), anti-phospho-FOXO1 (Cell Signaling Technology, 9464, 1:500), anti-FOXO1 (Cell Signaling Technology, 2880, 1:500) and anti-Histone-3 (Cell Signaling Technology, 9715, 1:2000).

Total protein was isolated with NucleoSpin TriPrep kit (Macherey-Nagel) from 3 × 10⁶ PMNs. Proteins from the nuclear and cytoplasmic fractions were isolated by using the Nuclear and Cytoplasmic Protein Extraction Kit (Thermo Scientific). Western blotting was performed by using AnykD Mini-PROTEAN TGX Gels (Biorad, Hercules, CA, USA) and nylon/nitrocellulose membranes (Biorad). Primary and secondary antibodies used were: rabbit anti-PAD4 (Abcam), rabbit anti-MPO (Cell Signalling Technologies, Beverly, MA, USA), mouse anti-β-Actin (Sigma-Aldrich), goat anti-Mouse and/or anti-Rabbit, human anti-HRP (Southern Biotech). HRP activity was detected by using SuperSignal West Pico Chemiluminescent Substrate (Thermo Scientific). Equal loading was verified by using beta-actin or histone H3, when appropriate. Western blots of citrullinated H3 (citH3) protein were performed according to Shechter et al.[25 (link)]. Densitometric analysis and protein quantification of the Western blots was performed by using the ImageJ software.

RIPA buffer supplemented with protease inhibitors was used to extract total protein; protein quantitation was conducted using the BCA protein assay kit (Biosharp, China). Samples were electrophoresed on a 10% SDS polyacrylamide gel (SDS-PAGE), then were transferred onto an NC membrane. The membrane was then blocked with 5% non-fat milk for 1 h, followed by washing, incubation with primary and secondary antibodies, and finally detected with enhanced chemiluminescence. The Nuclear and Cytoplasmic Protein Extraction Kit (78833, Thermo Scientific) was used for nuclear Nrf2 and cytosol Nrf2 extraction according to the manufacturer’s instructions, GAPDH and histone H3 (1:3000; Abcam) were served as internal reference proteins.

Total protein was extracted from normal and mastitis tissue using the Nuclear and Cytoplasmic Protein Extraction Kit (thermo) according to the manufacturer’s instructions. Protein was transferred onto polyvinylidene fluoride membranes and blocked overnight in 5% BSA in Tris-buffered saline with Tween 20 at 4°C. Membranes were incubated for 2 h at room temperature with the appropriate primary antibody. After three 15-min washes in Tris-buffered saline with Tween20, membranes were incubated with an appropriate secondary antibody conjugated to horseradish peroxidise diluted 1:1000 in block solution for 2 h. After three washes, blots were developed with ECL substrate using the Western Blotting Detection System (Amersham Life Science,UK).