Ab77612

OCCM-30 cells were cultured overnight on sterile Falcon™ Chambered Cell Culture Slides (354108, Fisher Scientific) and further fixed with 4% paraformaldehyde (30525-89-4, Sigma-Aldrich) for 10 min at room temperature. Cells were permeabilized with 0.5% Triton™ X-100 Surfact-Amps™ Detergent Solution (28313, Thermo-Fisher) for 20 min. Then, cells were incubated in blocking buffer containing 10% goat serum, 0.3 M glycine, 1% BSA (071M8410, Sigma) and 0.1% Tween-20 (P1379, Sigma-Aldrich) for 30 min at room temperature and further incubated with primary antibodies AdipoR1 (ab70362, Abcam) (dilution 1:250) or AdipoR2 (ab77612, Abcam) (dilution 1:250) at 4°C overnight. The secondary antibodies DyLight 488 polyclonal goat anti-rabbit (ab96899, Abcam) (dilution 1:500) or donkey anti-goat Alexa Fluor 647 (ab150131, Abcam) (dilution 1:500) conjugated to fluorescein isothiocyanate were used. After washing with 1× phosphate-buffered saline (PBS) (10010023, Thermo-Fisher), samples were mounted using a fluorescent Mounting Medium with DAPI (ab104139, Abcam). Staining was analyzed using a high-resolution fluorescence microscope (Leica Microsystems, Wetzlar, Germany) and photographed.

Lung tissue (50 mg) was added with 0.5 mL of the protein lysate (Cell Signaling Technology, Danvers, MA, USA), followed by sonication and homogenization. Then, samples were centrifuged at 10,000 × g for 10 min at 4°C to extract the supernatant for the determination of protein concentration (10 µg/µL) and western blotting. Electrophoresis, membrane transfer, and sealing were performed according to conventional procedures. Primary antibodies, including anti-adiponectin receptor 1 (AdipoR1) (ab126611, Abcam, Cambridge, UK), anti-AdipoR2 (ab77612, Abcam), and anti-T-cadherin (ab167407, Abcam), were diluted 1,000 times and incubated overnight. Anti-β-actin antibody (1:1,000, ab8226, Abcam) was used as an internal reference. Then, the membranes were incubated with the secondary antibody at a dilution of 1:10,000. The membranes were placed on a gel imager (Bio-Rad, Hercules, CA, USA) for development and gray-value calculations.