β actin

Total protein was extracted from lung tissues and BEAS-2B cells by protein extraction kit (Wanleibio). The concentration of protein was detected by BCA kit (Wanleibio). The protein was separated by SDS-PAGE and transferred to PVDF membrane (Millipore). The membrane was blocked by non-fat milk and then incubated with primary antibodies for SLC27A3 (Proteintech), STAU1 (Affinity), ACPL2 (Bioss), RABL4 (Biorbyt), and β-actin (Wanleibio), respectively. After 12-h of incubation at 4°C, the membrane was washed, incubated by the secondary antibody, and visualized by CEL (Wanleibio). β-actin serves as an internal control.

The detailed procedure of western blot was described briefly here. After different treatments, cells were lysed with RIPA buffer and the lysate was quantified with the BCA kit according to manufacturer instruction. A total of 30 μg protein of each group was loaded for electrophoresis in 10% SDS-PAGE gel. Then, protein was transferred onto PVDF membrane followed by blocking with 5% skim milk in TBST for 30 min. Primary antibodies MMP-3 (Abcam, USA) and β-actin (Wanlei, China) were diluted into 5% BSA dissolved in TBST at 1 : 1000 based on the datasheets of antibodies. PVDF membranes were incubated with diluted primary antibodies overnight at 4°C. Secondary antibodies were diluted into 5% skim milk dissolved in TBST (1 : 10000) and incubated with membranes for 1 h to 1.5 h at room temperature with gentle shaking. Enhanced chemiluminescence (ECL) system was employed for imaging.

Western blotting was carried out according to our previously described protocol [31 (link)] and described briefly as follows. Kidney tissue or NRK-52E cells were extracted by radioimmunoprecipitation (RIPA) (Wanlei Biotechnology Co. Ltd, Shenyang, China, Catalogue No.: WLA016a) and measured by a BCA assay according to the manufacturer's instructions (Wanlei Biotechnology Co. Ltd, Shenyang, China, Catalogue No.: WLA004b). Proteins were separated in SDS-PAGE gels and transferred to polyvinylidene fluoride (PVDF) membranes. Rabbit-anti-mouse primary antibodies were used to bind target proteins including PPAR-γ (Santa Cruz Biotechnology, Inc., Dallas, Texas, USA, Catalogue No.: sc-390740), GLP-1R (Bioss, Beijing, China, Catalogue No.: bs-1559R), and β-actin (Wanlei biotechnology co. Ltd, Shenyang, China, Catalogue No.: WL01845) at 4°C overnight. And then, goat-anti-rabbit secondary antibody conjugated with HRP (Wanlei Biotechnology Co. Ltd, Shenyang, China, Catalogue No.: WLA023a) incubation was performed for binding primary antibodies. The ECL kit (Wanlei Biotechnology Co. Ltd, Shenyang, China, Catalogue No.: WLA006a) was utilized before exposure to detect the protein levels.

Western blot was used to detect the expression of protein. Membranes were blocked with 5% TBST diluted nonfat powdered milk at room temperature for 1 hour incubated with primary antibody (HMGB1, sc-56,698, santa cruz; TLR4S, sc-293,072 santa cruz; Phosphorylation of NF(P-NF)-κB p65, sc-166,748 santa cruz; Cleaved caspase3, #9961, Cell Signaling Technology; NF-κB p65 #8242, Cell Signaling Technology; bcl-2, WL01556, Wanlei; Bax WL01637 Wanlei; GAPDH WL01114; β-actin WL01372 Wanlei.) at 4 C for 14 h. β-actin and GAPDH were used as the standard proteins. The secondary antibody was diluted with blocking solution and incubated membranes for 90 mins at room temperature [29 (link)]. Image J was used to analyze the gray value of the protein band.

Antibodies targeting the following proteins were used in this study: cyclin D1 (WL01435a), CDK2 (WL01543), p27 (WL04174), cleaved caspase-9 (WL01838), cleaved caspase-3 (WL01992), Bcl-2 (WL01556), Bax (WL01637), LC3BII/I (WL01506), beclin-1 (WL02508), p62 (WL02385), p-JNK (WL01813), JNK (WL01295), c-JUN (WL02863), E-cadherin (WL01482), vimentin (WL01960), MMP-2 (WL1579), MMP-9 (WL01580), β-actin (WL01372), goat anti-rabbit IgG-HRP (WLA023) (all from Wanleibio, China), and p-c-JUN (AF3095, Affinity, United States). Purified peiminine (98%) was obtained from Yuanye Bio Co., Ltd (Shanghai, China; B20081). Cell Counting Kit-8 (CCK-8; C0037), the live/dead assay kit, the cell cycle and apoptosis analysis kit, the ROS Kit, NAC, SP600125, and the terminal dUTP nick-end labeling (TUNEL) Kit (C1086) were acquired from Beyotime Institute of Biotechnology (Shanghai, China). Tetramethylrhodamine methyl ester (TMRM; I34361) and Hoechst 3342 (H3570) were sourced from Life Technology (OR, United States). The mRFP-GFP-LC3 adenovirus was acquired from HanBio Technology Co., Ltd (Shanghai, China). The rest of the reagents and experimental materials were purchased from common commercial sources.