Anti adipor1

An equal amount of protein (30 μg) was analyzed per sample using SDS polyacrylamide gel electrophoresis. Separated proteins were then transferred to a PVDF membrane. The membrane was incubated with primary antibodies overnight at 4°C with gentle shaking. The membranes were washed and incubated with a secondary antibody, followed by electrochemical luminescence detection. Relative protein levels were quantified by scanning densitometry and analyzed using ImageJ software. Protein detection was carried out using anti-GAPDH, anti-β-actin, anti-HIF-1α, anti-IL-17A, anti-GLUT1, anti-HK2 (proteintech) and anti-AdipoR1 (Abcam) Abs.

Western blot analysis was employed to detect the protein expression level of TGF-β₁, CTGF, and α-SMA in lung tissue, and collagen type III, α-SMA, and AdipoR1/R2 in human lung WI-38 fibroblasts. Whole lung tissue homogenate or WI-38 lung fibroblasts lysates were prepared via lysis buffer [TianGen Biotech (Beijing) Co., Ltd.]. The lung tissue homogenate or cell lysates were separated by electrophoresis on 10% SDS-PAGE. Gels were transferred to a nitrocellulose membrane, blocked in 5% nonfat dry milk diluted in Tris-buffered saline. Blots were incubated overnight with anti-TGF-β_1, anti-CTGF, anti-α-SMA, anti-collagen type III, anti-AdipoR1, or anti-AdipoR2 antibodies (all from Abcam), at 4°C. Washed blots were incubated at room temperature for 60 minutes with a secondary biotinylated antibody (Zhongshan Golden Bridge Bio-technology, Beijing). After three 10 minute washings with TBST, membranes were analyzed by Western (Bio-Rad Laboratories Inc., Hercules, CA). Sample loadings were normalized by Western blot analysis with anti-GAPDH (Abcam).

The HUVEC cells were fixed, permeabilized and subsequently combined with rabbit anti-adipoR1, anti-adipoR2 or anti-T-cadherin antibodies from Abcam (UK). Then, the cells were incubated with FITC-conjugated anti-IgG antibodies (Sigma, USA) in the dark. The cells were mounted and observed on a fluorescence microscope (Olympus, USA).