Adipor2

Total protein was isolated from heart tissues, NRCMs and macrophages and then subjected to SDS-PAGE (50 μg per sample). After transfer onto Immobilon membranes (Millipore, Billerica, MA, USA), proteins were incubated overnight at 4°C with primary antibodies against CTRP1, adiponectin R1 (Adipo R1), and Adipo R2 purchased from Abcam (1:1000 dilution) and TLR4 and GAPDH purchased from (Cell Signaling Technology (1:1000 dilution). Blots were developed with enhanced chemiluminescence (ECL) reagents (Bio-Rad, Hercules, CA, USA) and captured by a ChemiDoc MP Imaging System (Bio-Rad). GAPDH served as an internal reference protein.
Bone marrow-derived macrophages were cotransfected with psicoR-HA-TLR4 and psicoRFlag-CTRP1 or psicoRFlag-Adipo R1. The macrophage lysates were treated with a Protein G Plus/Protein A agarose suspension (Santa Cruz, CA, USA) and then incubated with antibodies against HA or Flag (Proteintech, USA). An agarose suspension of Protein G Plus/Protein A was added once again, and immunoprecipitants were subjected to SDS-PAGE electrophoresis.

The BV2 cells were washed with phosphate-buffered saline (PBS) and collected. Cell pellets were lysed with ice-cold RIPA buffer (Sigma-Aldrich). The lysates were centrifuged at 15,900 g for 30 min at 4°C to produce whole-cell extracts. Protein (30 μg) in cells was separated on a 10% SDS-polyacrylamide gel and transferred onto a polyvinylidene difluoride membrane. After blocking with 5% skim milk prepared in Tris-buffered saline-Tween (20 nM Tris [pH 7.2], 150 mM NaCl, 0.1% Tween 20) for 1 h, 30 min at room temperature, immunoblots were incubated for 18 h at 4°C with primary antibodies that detect AdipoR1 (1:1000, Abcam, Cambridge, MA, USA), AdipoR2 (1:1000, Abcam, Cambridge, MA, USA), CD86 (1:1000, Cell Signaling, Danvers, MA, USA), p-NF-κB (1:1000, Cell Signaling), PPAR-γ (1:1000, Cell Signaling, Danvers, MA), p-PPAR-γ (1:1000, Santa Cruz Biotechnology, Santa Cruz, CA, USA) and β-actin (1:1000; Millipore, Billerica, MA, USA). All blots were then incubated with appropriate secondary antibodies (Abcam, Cambridge, MA, USA) for 1 h 30 min at room temperature. All blots were visualized using ECL solution (Millipore, Billerica, MA, USA).

bEnd.3 cells were washed with PBS and collected. Cell pellets were lysed with ice-cold RIPA buffer (Sigma-Aldrich). The lysates were centrifuged at 13 000 rpm for 30 min at 4 °C to produce whole-cell extracts. Protein (30 μg) in cells was separated on a 12% SDS-polyacrylamide gel and transferred onto a polyvinylidene difluoride membrane. After blocking with skimmed milk prepared in Tris-buffered saline-tween (TBST) (20 nM Tris (pH 7.2), 150 mM NaCl, 0.1% Tween 20) for 1 h at room temperature, immunoblots were incubated for 16 h at 4 °C with primary antibodies that detect p-NF-κB (1:1000, Cell Signaling, Danvers, MA, USA), NF-κB (1:1000, Cell Signaling), AdipoR1 (1:1000, Abcam, Cambridge, MA, USA), AdipoR2 (1:1000, Abcam), Claudin 5 (1:1000, Cell Signaling), CD31 (1:1000, Abcam) or β-actin (1:1000; Millipore, Billerica, MA, USA). Blots were then incubated with each secondary antibody (Abcam) for 1 h and 30 min at room temperature. Blots were visualized by ECL solution (Millipore).