Anti cpt1a

For immunofluorescence analysis of NRK52E cells, cells on glass coverslips were fixed with ice-cold methanol for 10 min and then permeabilized with 0.1% Triton for 5 min at 25°C. The cells were blocked with 5% bovine serum albumin (BSA, Biosharp, Beijing, China), incubated overnight at 4°C with primary antibodies (anti-Cpt1a, 1:100, Abclonal, Wuhan, China), and then incubated in the dark with secondary antibodies (DyLight 488 goat anti-rabbit IgG, 1:500, Abbkine, Wuhan, China) for 1 h at 25°C. Finally, nuclei were stained with Hoechst (Beyotime) for 5 min.
Immunofluorescence analysis of the left kidney tissue was performed on paraffin-embedded sections mounted on glass slides. Primary antibody anti-F4/80 (1:1000, Cell Signaling Technology) and HRP-goat anti-rabbit IgG secondary antibody (1:4000, Abcam) were used. The slides were visualized under a fluorescence microscope (Nikon Eclipse, Tokyo, Japan).

Proteins from kidney tissues and cultured cells were extracted with RIPA lysis buffer (Beyotime). Protease and phosphatase inhibitor cocktails were added for the extraction of phosphorylated proteins. The total protein concentration was measured using a BCA protein assay kit (Beyotime). Proteins (40–80 μg) were separated using SDS-PAGE (10%) and transferred to a PVDF membrane. The membranes were blocked with 5% BSA for 1.5 h and incubated with primary antibodies overnight at 4°C, followed by secondary antibodies (goat anti-rabbit, 1:3000; anti-mouse, 1:5000; Servicebio, Wuhan, China) for 1.5 h. The following primary antibodies were used: anti-α-SMA (1:3000, Proteintech, Wuhan, China), anti-Col1a1 (1:1000, Cell Signaling Technology), anti-collagen type III alpha 1 chain (anti-Col3a1, 1:1000, Abclonal), anti-Cpt1a (1:1000, Abclonal), anti-Acox1 (1:1000, Abclonal), anti-phosphorylation-AMP-activated protein kinase (anti-p-AMPK, 1:1000, Abclonal), and anti-glyceraldehyde-phosphate dehydrogenase (anti-GAPDH, 1:50000, Abclonal). Images were visualized using a Gene Gnome XRQ system (Syngene, Cambridge, UK).