Ecl plus western blotting detection reagent

Protein preparation and western blot analysis were performed as described previously (Hu et al., 2007 (link)). Briefly, samples were separated by SDS-PAGE and transferred to PVDF membranes (Bio-Rad, Hercules, CA, United States). The blots were blocked with non-fat dry milk (7.5%) in Tris-buffered saline/Tween 20 (TBST) for 2 h and then incubated with primary antibodies at 4°C overnight. Antibodies against p-CREB Ser133 (#9198), CREB (#9197), ERK1/2 (#4695), p-ERK1/2 Thr202/Tyr204 (#4370), p-STAT3 Tyr705 (#9145), STAT3 (#4904), and GAPDH (#5174) were purchased from Cell Signaling Technology (Danvers, MA, United States) and diluted at 1:1000 for use. The horseradish peroxidase-conjugated secondary antibodies (Dingguo, Beijing, China) were used at 1:20,000 dilutions for GAPDH and 1:5000 otherwise. Immunoreactive proteins were detected by ECL plus Western blotting detection reagent (GE Healthcare, Buckinghamshire, United Kingdom) and quantified by densitometry (Bio-Rad).

Differentiated 3T3-L1 adipocytes co-cultured with RAW264.7 cells were treated with MNG and lysed using RIPA buffer (Cell Signaling, Danvers, MA, USA). Equal amounts of protein extracts (20 μg) were separated on a 10% sodium dodecyl sulfate-polyacrylamide gel and transferred onto polyvinylidene difluoride membranes. The membranes were incubated with primary antibodies (1:1000; Cell Signaling Technology, Inc., Danvers, MA, USA) against C/EBPα, C/EBPβ, PPARγ, COX-2, and iNOS overnight at 4 °C, followed by incubation with secondary antibodies (1:2000; Cell Signaling Technology, Inc.). ECL Plus Western blotting detection reagent (GE Healthcare, Little Chalfont, UK) was used, and the signals were visualized using a chemiluminescence system (FUSION Solo, PEQLAB Biotechnologie GmbH, Erlangen, Germany).

Prepared cell extracts from different groups were incubated with 1 μg anti‐MD2 (R&D system) or anti‐TLR4 (ab8376, Abcam, UK) for 1 hour at 4°C, and immunoprecipitation was made with protein A Agarose Beads (9863T, CST, USA) at 4°C overnight. Finally, samples were immunoblotted for detection of TLR4 or MyD88 as co‐precipitated protein.
Myocardial tissues or cells were homogenized with ice‐cold RIPA lysate (Beijing Applygen Technology Inc, China) containing 1% protease inhibitor and 2% protein phosphatase inhibitor (Beijing Applygen Technology Inc, China) and then centrifuged at 12 000 g for 10 minutes at 4°C. Bicinchoninic Acid (BCA) Protein Assay Kit (Beijing Applygen Technology Inc, China) was used to measure the protein concentration. Quantitative samples were separated on 10% SDS‐PAGE gels and transferred to polyvinylidene fluoride (PVDF) membranes. The membranes were blocked with 5% skim milk in 1 x Tris‐buffered saline for 2 hours, followed by incubation with the primary antibodies at 4°C overnight and secondary antibody at room temperature for 1 hour. The membranes were exposed to ECL (ECL Plus Western blotting Detection Reagent, GE Healthcare, United States) in the darkroom for about 10 seconds. Density of bands was quantified by ImageJ. The antibodies we used are listed in Table 2.

Input, Nu, and Cyt aliquots of three independent experiments were thawed, loaded completely in BioRad pre-cast 4–20% Mini-PROTEAN TGX Stain-Free Protein Gels (10 wells), and run at 150 V constant. Transference to a 0.45-μm nitrocellulose membrane (BioRad) was done at 300 mA constant for 1 h 30′ in a cold room in 20% methanol transfer buffer. The membrane was blocked with 4% milk in TBS with 0.5% Tween-20 for 1 h at room temperature and incubated at 4 °C overnight with the following primary antibody solutions (4% milk, 0.5% Tween-TBS): anti-p120 (Ctnnd1) 1:2000 (Millipore 05-1567, clone 15D2); anti-Mbnl1 1:100 (DSHB-MB2a(3b4)), and anti-Ac H3 1:1000 (Millipore 06-599). The membranes were incubated for 1 h at room temperature with secondary antibodies in 4% milk, 0.5% Tween-TBS: anti-mouse HRP (Life A16072) 1:10,000; anti-rabbit HRP (Thermo 31460) 1:10,000. Signal detection was performed with an enhanced chemiluminescence kit (ECL Plus Western blotting detection reagent from GE Healthcare, Piscataway Township, NJ, USA), and bands were detected by film exposition. After the first of detections, the membranes were quenched in 0.05% sodium azide in 0.5% Tween-TBS for 30 min with gentle rocking, washed three times with 0.5% Tween-TBS, and hybridized with anti-tubulin coupled HRP (Thermo MA5-16308-HRP) to produce cytoplasmic loading controls.

Protein lysates were separated by 4–20% Tris-HCl SDS-PAGE. Gel transfer to a nitrocellulose membrane was conducted using the iBlot® gel transfer system (Invitrogen). Membranes were blocked and incubated with primary antibody, followed by incubation in HRP conjugated secondary antibody (GE Healthcare, Little Chalfont, UK). Membrane was treated with ECL Plus Western Blotting Detection reagent (GE Healthcare) and visualized on Amersham Hyperfilm™ ECL (GE Healthcare). Quantification of the bands was performed using the Quantity One 1-D Analysis Software (Bio-Rad Laboratories/Life Science Research, Hercules, CA), and the number corresponding to the expression relative to the β-actin band are displayed below each band.