Enhanced chemiluminescence ecl kit

Tumors harvested were homogenized in a lysis buffer. Total protein and mRNA extracts were obtained using RIPA (Sigma-Aldrich) and RNAiso Plus (Takara) according to manufacturer’s protocols, respectively. qRT-PCR was used to measure mRNA levels. First, extracted total RNA was reverse-transcribed into cDNA. Then, the cDNA was subjected to qRT-PCR (CFX96T Real-time System, Bio-Rad) and amplified according to following conditions: 40 cycles including denaturation at 94°C for 1 min; annealing: 60°C for 1 min [38 (link)]; extension: 72°C for 1 min. For Western blot assay, 20 μg of total protein was subjected to electrophoresis on a sodium dodecyl sulfate polyacrylamide gel (90V for spacer gel, 120V for separation gel) and then transferred to a polyvinylidene fluoride (PVDF) membrane. The blotted membranes were incubated with specific primary antibodies overnight, and subsequently, incubated with secondary horseradish peroxidase (HRP)-labeled antibody for 4 h. After washing for 3 times, immunostaining was performed using an enhanced chemiluminescence (ECL) kit (GE Healthcare, UK) and then imaged on a Biospectrum600 Imaging System (Upland, CA, USA [39 (link)]).

Dulbecco’s Modified Eagle Medium (DMEM) was purchased from Thermo Fisher (Beijing, China). Fetal bovine serum (FBS) and antibiotics (streptomycin and penicillin) were obtained from GIBCO (New York, NY, USA). All tissue culture plasticware was obtained from Corning (Corning, NY, USA). ISL (purity: 98.26%) was purchased from Shaanxi Green Bio-Engineering Co., Ltd. (Xi’an, China), and dissolved in 0.5% sodium carboxymethyl cellulose (CMC-Na). MTT, LPS, and oil red O were all from Sigma-Aldrich (St. Louis, MO, USA). LDL, 1 mg/mL, was obtained from Prospect (Ness Ziona, Israel) and reacted with 10 μmol/L copper sulfate for 24 h at 37 °C. After dialysis, the LDL was filtered with a 0.22 μm membrane resulting in ox-LDL; the concentration was determined using the Lowry method. Anti-CD36 and anti-ABCA1 monoclonal antibodies were obtained from Abcam (Cambridge, MA, USA). Anti-PPARγ polyclonal antibody and anti-β-actin monoclonal antibody were from Santa Cruz Biotechnology (Delaware Ave Santa Cruz, CA, USA). Horseradish peroxidase (HRP)-conjugated secondary antibodies were from Jackson Laboratory (Main St Bar Harbor, ME, USA). The enhanced chemiluminescence (ECL) kit was purchased from GE Healthcare (Waukesha, WI, USA). PCR primers were from Sangon Biotech (Shanghai, China). All other chemicals were of the best grade and obtained from commercial sources.

For Western blot analysis, the cells were lysed in 25 μl Nonidet P-40 buffer (1 %) before 25 μl reducing loading buffer was added. After incubation for 4 min at 95 °C, the lysates were loaded on 10 % SDS gels for electrophoresis at 40 to 100 V for approximately 3 h. Then the proteins were blotted onto a nitrocellulose membrane (Roth, Karlsruhe, Germany) for 1 h using a semi-dry blotting technique (1 mA/cm²). The membrane was blocked in Tris-buffered saline with 0.1 % Tween-20 (TBS-T) with 5 % (w/v) non-fat dry milk for 1 h, washed, and then incubated with a biotinylated anti-MICA Ab (0.4 μg/ml, polyclonal goat IgG, BAF1300, R&D Systems) and an anti-β-actin mAb (1:10,000, mouse IgG₁, clone AC-15, Sigma-Aldrich) in TBS-T together with 5 % (w/v) non-fat dry milk overnight at 4 °C. After being washed three times for 10 min in TBS-T, the membrane was incubated with horseradish peroxidase (HRP)-conjugated streptavidin (1:2000, BioLegend, Fell, Germany) and HRP-labeled goat-anti-mouse IgG secondary Ab (1:10,000, 115-035-003, Jackson Laboratories, via Dianova, Hamburg, Germany). Detection was done using an enhanced chemiluminescence (ECL) kit (GE Healthcare), and chemiluminescence was measured using a digital image acquisition system (Intas Chemilux Entry, Intas, Göttingen, Germany).

Cell lysates (10–20 μg) were resolved in 15% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto PVDF membranes, which were then incubated with either the appropriate antibodies overnight at 4 °C or biotin-structured aptamer for 1 h at 37 °C. After washing, blots were incubated with the corresponding HRP-conjugated secondary antibody (GE Healthcare, Madrid, Spain) or HRP-streptavidin (Sigma Aldrich, Madrid, Spain) for 1 h at room temperature. Finally, the membranes were developed with either the enhanced chemiluminescence (ECL) kit (GE Healthcare, Madrid, Spain) or Clarity Western ECL Substrate (BioRad, Barcelona, Spain). PageRuler Plus Prestained Protein Ladder (Thermo Scientific, Massachusetts, USA) was used in all of the experiments. β-Actin (Sigma, St. Louis, MO, USA) and α-histone H4 (Abcam, Cambridge, UK) antibodies were used as loading controls. Supplementary Table S2 shows the antibodies used in this study.