Klotho

The hypothalamus and distal ileum were collected 8 weeks postsurgery and lysed in radioimmunoprecipitation assay buffer. Protein concentrations of the supernatant were quantified using the BCA Protein Assay Kit (Solarbio Life Science, Beijing, P. R. China), and equivalent amounts of protein were subjected to 10% sodium dodecyl sulfate−polyacrylamide gel electrophoresis and transferred to a 0.2 μm aperture polyvinylidene fluoride membrane. The membranes were blocked in 5% bovine serum albumin liquid and incubated with primary TGR5 (Abcam, Cambridge, UK), FXR (ABclonal, Wuhan, P.R. China), MC4R (ABclonal, Wuhan, P.R. China), Klotho (Proteintech, Wuhan, P.R. China), POMC (Wuhan, P.R. China) and β-actin (Cell Signaling Technology, Danvers, MA, USA) antibodies at 4 °C overnight. The membranes were washed three times with Tris-buffered saline and Tween 20, goat anti-rabbit (ABclonal, Wuhan, P.R. China) and goat anti-mouse (ABclonal, Wuhan, P.R. China) were incubated at room temperature for 1 hour with shaking. After three rinses with TBST solution, the membrane was scanned. The relative concentration of protein was quantified by densitometry using the Tanon Imaging System and ImageJ software.

By homogenization and lysis in RIPA Lysis Buffer (Beyotime), we extracted total proteins from lung tissues, primary ATII cells, and A549 cells. The protein content was quantified using a BCA kit (Beyotime). Western Blot analysis in lung tissues and cells was performed against DRP1, phosphorylated-DRP1 (p-DRP1) (Ser616), MFF, OPA1, MFN2 (1:1000, Cell Signaling Technology, Danvers, MA, USA), PINK1, PARK2, SQSTM1/p62, LC3b, P16, H2AX (1:1000, Abcam Cambridge, MA, USA), Klotho and GAPDH (1:1000, Proteintech, Wuhan, Hubei, China). Bands were developed by ECL chemiluminescent substrate (Millipore, Billerica, MA, USA).

All collected proteins were added to sodium dodecyl sulfate (SDS) sample buffer (62.5 mM Tris (pH 6.7), 1.25% SDS, 12.5% glycerol, and 2.5% β-mercaptoethanol). Proteins and the prestained protein marker (10–315 kDa) (TD-PM10315, BIOTOOLS Co., Ltd., Taipei, Taiwan) were loaded into an SDS-PAGE gel. A PVDF membrane containing transferred proteins was incubated with 5% nonfat milk with primary antibodies anti-microtubule-associated protein 1A/1B-light chain 3 (LC3) (Cell Signaling, Beverly, MA), collagen 1 (Proteintech, Rosemont IL), autophagy-related 5 (ATG5) (Proteintech), CTGF (Proteintech), PAI-1 (Cell Signaling), SIRT1 (ABclonal Inc., Woburn, MA), caspase-3 (ABclonal Inc.), Hsp27 (ABclonal Inc.), BAX (ABclonal Inc.), Klotho (Proteintech), p53 (Proteintech), p21(Proteintech), p16 (Proteintech), G6PD (Proteintech), and GAPDH (Proteintech). After the hybridization process with the abovementioned antibodies on the PVDF membrane, the membrane was rinsed with TBS-T for 15 min three times. Subsequently, the PVDF membrane was further treated with anti-mouse (Jackson) or anti-rabbit (Jackson) secondary antibody for 2 h and rinsed with TBS-T for 15 min over three times. The protein bands of the PVDF membrane were visible by performing an enhanced chemiluminescence system (Amersham, Little Chalfont, United Kingdom).