Dpp 4 assay kit

To measure the activity of DPP-4, we used a DPP-4 Assay Kit (Enzo Life Sciences, Farmingdale, NY, USA) with the Gly-Pro-para-nitroaniline (pNA) chromogenic substrate, according to the manufacturer’s instructions. Cells grown in six-well plates were homogenated in buffer containing 200 µL ordinary RIPA lysis (Solarbio, Beijing, China), 0.2 µL PMSF (Solarbio, Beijing, China), and 0.2 µL protease inhibitor cocktail (Boster Biological Technology, Wuhan, China). Start the assay by mixing 20 µL of lysate with 10 µL of 5 mM Gly-Pro-pNA and then read the plate record data immediately at 2 min intervals for a total of 60 min. DPP-4 activity was calculated according to the released pNA by measuring the absorbance at 405 nM with a standard curve of p-nitroanalide. The enzyme activity was defined as the amount of enzyme catalyzing the formation of p-nitroanalide/s.

The primary antibodies used in this study targeted SDF-1α (Novus Biologicals, Littleton, CO, United States), DPP-4 (Abcam, Cambridge, MA, United States), p-PKA (clone EP2606Y, Abcam, Cambridge, MA, United States), PKA (Cell Signaling, Danvers, MA, United States), NOX2 (clone EPR6991, Abcam, Cambridge, MA, United States), p-ERK1/2 (clone D13.14.4E, Cell Signaling, Danvers, MA, United States), ERK1/2 (Cell Signaling, Danvers, MA, United States), transforming growth factor-β1 (TGF-β1) (clone EPR18163, Abcam, Cambridge, MA, United States), podocin (Abcam, Cambridge, MA, United States), desmin (Abcam, Cambridge, MA, United States), nephrin (clone B-12, Santa Cruz Biotechnology Inc., Dallas, TX, United States), fibronectin (Proteintech, Rosemont, IL, United States), Fsp1 (clone EPR14639(2), Abcam, Cambridge, MA, United States), and α-SMA (clone 1A4, Abcam, Cambridge, MA, United States). ROS levels in renal tissue were tested with a dihydroethidium (DHE) assay kit (Beyotime, Jiangsu, China), and ROS levels in cells were examined with a DCFH-DA assay kit (Beyotime, Jiangsu, China). DPP-4 enzyme activity was tested using a DPP-4 assay kit (Enzo Life Sciences, Farmingdale, NY, United States).