Nuclear extraction kit

HT29 and HT29‐MTX cells were grown in 75 cm² cell culture flasks as described previously until confluent. Cell were treated for 48 h with either 50 µM AA, EPA, DHA or equivalent ethanol (control) in duplicate and removed with 6 mL 0.05% v/v Trypsin 0.53 mmol/L 53 mM EDTA.4Na (Invitrogen). Nuclear extracts were isolated using a “Nuclear Extraction Kit” (Cayman Chemical, Catalog No. 10009277) and samples were tested for protein concentration using BCA Protein Assay Kit (Pierce). Nuclear extracts were added to the PPARα, δ, γ ‘Complete Transcription Factor’ Assay plate (Cayman Chemical, Catalog No. 10008878) using each biological replicate and testing for each PPAR following manufacturer's instructions. Sample optical density (450 nm) was corrected for protein concentration.

Nuclear and cytosolic proteins were fractionated using a Nuclear Extraction Kit (Cayman Chemicals, Ann Arbor, MI) according to the manufacturer’s instructions. Briefly, cells were harvested and centrifuged (3000 rpm, 5 min) at 4 °C. The cell pellets were mixed with hypotonic buffer containing a phosphatase inhibitor and a protease inhibitor. After 10 min of incubation on ice, the cells were treated with 10% Nonidet P40 Assay Reagent. Nuclei were recovered by centrifugation (14,000 rpm, 30 s), and the supernatant was stored as a cytoplasmic extract at −80°C until use. The nuclei were extracted with Nuclear Extraction Buffer for 30 min on ice. Insoluble material was removed by centrifugation (14,000 rpm, 10 min). Finally, the supernatant was used as a nuclear extract.

Following drug treatment, floating cells and attached cells were collected and centrifuged at 1000 rpm for 5 min at 4^oC. The resultant pellet was lysed with RIPA lysis buffer containing protease and phosphatase inhibitor cocktail and centrifuged at 12,000 rpm for 10 min at 4^oC. Supernatants were then collected and total protein was determined by BioRad reagent (BioRad Laboratories, Hercules, CA). Where applicable, cytoplasmic and nuclear fractions were separated by use of a nuclear extraction kit according to manufacturer's instructions (Cayman Chemical, Ann Arbor, MI). Fifty micrograms of protein were resolved in SDS-polyacrylamide gels (SDS-PAGE) and transferred onto nitrocellulose membranes (BioRad Laboratories, Hercules, CA). Membranes were next blocked with 5% nonfat milk followed by incubation with antibodies against FADD, cleaved caspase-3, pIκB, p50, p65, Lamin B or beta-actin. Membranes were next washed and incubated with appropriate secondary antibody conjugated to HRP (GE Healthcare Life Sciences, Piscataway, NJ). Following secondary antibody incubation, membranes were washed and signal detected with ECL detection reagent (GE Healthcare Life Sciences, Piscataway, NJ). Lamin B served as a nuclear marker. Beta-actin served as a protein loading control.

A nuclear extraction kit (Cayman, Ann Arbor, MI, USA) was used to separate cytosolic and nuclear fractions. Each extracted fraction was lysed according to the protocol provided by the manufacturer.

3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT), minimum essential medium (MEM), IFNγ, PMA, sodium dodecyl sulfate (SDS), and bovine serum albumin (BSA), were purchased from Sigma (St. Louis, MO, USA). All CGA isomers, (#1 to #6) were obtained from the Cerillian Corporation (Round Rock, TX, USA) and Chengdu Must Bio-Technology Co. (Chengdu, Sichuan, China). Fetal bovine serum (FBS), penicillin, and streptomycin were purchased from Gibco^® (Grand Island, NY, USA). The Nuclear Extraction Kit and p65 Transcription Factor Assay Kit was purchased from the Cayman Chemical Company (Ann Arbor, MI, USA).

Total cell lysates and nuclear extracts were prepared using ice cold RIPA buffer (Thermo Fisher Scientific, Inc., Rockford, IL) and Nuclear Extraction Kit (Cayman), respectively. The protein concentration was measured by the Bradford method (Bio-Rad Laboratories, Hercules, CA, USA) using bovine serum albumin as a standard. Cell lysates were separated on 8–12% SDS-PAGE and transferred onto Hybond ECL nitrocellulose (GE Healthcare) at 20 volt overnight at 4°C. The membranes were blocked at 4°C in PBST blocking buffer (5% BSA in PBS with 0.05% Tween 20, pH 7.4) for 8 h. Blots were analyzed with each antibody (Table 1) at a dilution of 1:1000 overnight at 4°C. After three washes with PBST, the blots were incubated with suitable horseradish peroxidase-conjugated secondary antibody (Jackson ImmunoResearch, West Grove, PA) at a dilution of 1:10,000–25,000 for 1 h. The blots were washed again and the proteins of interest were detected by Amersham ECL Prime Western Blotting Detection Reagents (GE Healthcare) according to the manufacturer’s instructions, and the chemiluminescence signal was then visualized with X-ray film. For reprobing, blots were treated with Restore stripping solution (Thermo Scientific). The intensities of these bands were analyzed with Phoretix Gel Analysis Software (Nonlinear Dynamics, Newcastle upon Tyne, UK).

Total protein was extracted from cells and tissues using the RIPA buffer (Solarbio, Beijing, China) and the cytoplasmic and nuclear proteins for the nuclear extraction kit (Cayman Chemical, Ann Arbor, MI) according to the manufacturer's protocol. Equal amounts of total protein were separated by 10% SDS‐PAGE and then transferred to PVDF membranes. The membranes were blocked with 5% non‐fat milk at room temperature for 1 hour and then incubated with the appropriate primary antibodies overnight at 4°C. The membranes were subsequently incubated with horseradish peroxidase‐conjugated antibodies and visualized with an enhanced chemiluminescence reagent (Millipore, Wanchai, Hong Kong).