Adipocyte fatty acid-binding protein oversecretion hinders intestinal stem cell differentiation and gut repair
Description
Uncropped western blot in Figures2,6 and 7
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Freshly scraped intestinal crypts, Drosophila whole intestine, or Drosophila hemolymph were lysed with RIPA Lysis Buffer (Beyotime Biotechnology, P0013B) containing an inhibitor cocktail (MedChemExpress, HY-K0010 and PMSF (MedChemExpress, HY-B0496), followed by homogenization on ice and incubation on ice for 30 minutes. Protein concentration was determined using the BCA protein assay kit (Beyotime Biotechnology, Shanghai, China). Equal amounts of protein were loaded and subjected to SDS-polyacrylamide gel electrophoresis. After membrane transfer, the membranes were blocked with 5% BSA at room temperature for one hour, followed by overnight incubation with primary antibodies: - Rabbit anti-ACTB (1:50000, ABclonal AC026) - Rabbit anti-α-Tubulin (1:2000, ABclonal A6830) - Mouse anti-Myc (1:1000, Beyotime Biotechnology AF0033) - Rabbit anti-HA (1:1000, Cell Signaling Technology 3724) - Rabbit anti-FLAG (1:2000, Beyotime Biotechnology AG8050) - Rabbit anti-PMP70 (1:500, Abcam ab3421) - Rabbit anti-ACOX1 (1:250, Proteintech 10957-1-AP) - Rabbit anti-active β-Catenin (1:1000, Cell Signaling Technology 19807S) - Rabbit anti-Fth1 (1:1000, ABclonal A1144) - Rabbit anti-β-Catenin (1:6000, Proteintech 51067-2-AP) - Mouse anti-PPARA (1:1000, Proteintech 66826-1-Ig) - Rabbit anti-PEX11A (1:1000, ABclonal A17576) - Rabbit anti-Notch1 (1:500, Abcam ab52301) For secondary antibodies, goat anti-rabbit IgG (1:2000, Thermo Fisher Scientific, A-11008) or goat anti-mouse IgG (1:2000, Thermo Fisher Scientific) conjugated to horseradish peroxidase (HRP, Servicebio) were used.
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Ministry of Science and Technology of the People's Republic of China
2020YFA0803602