HCEC lysates were prepared using RIPA buffer (r0010; Santa Cruz Biotechnology, Dallas, TX, USA) supplemented with 1% phenylmethylsulfonyl fluoride. A volume of 50 µL lysate was added into each well of a 24-well plate and incubated for 15 minutes on ice. The lysate was collected by scraping the cells with a 1 mL pipette tip and transferred to a 1.5 mL centrifuge tube. After centrifugation at 12,000 rpm for 20 minutes at 4°C, the supernatant was collected and mixed with 5 × loading buffer. The mixture was then heated at 95°C for 10 minutes. The sample was either directly used for Western blot or stored at −80°C for future use.
For Western blot analysis, protein samples were subjected to 10% acrylamide gel electrophoresis and transferred to polyvinylidene difluoride membranes for antibody incubation. Dusp5 antibody (1:1000, ab200708; Abcam) was used, and GAPDH antibody (1:1000, LK9002; Tianjin Sungene Biotech Co., Ltd, Tianjin, China) was used as a loading control. The protein bands were quantified by ImageJ grayscale image peak analysis, which involved selecting the bands with the rectangle tool after the images were inverted to the 8-bit format.
For Western blot analysis, protein samples were subjected to 10% acrylamide gel electrophoresis and transferred to polyvinylidene difluoride membranes for antibody incubation. Dusp5 antibody (1:1000, ab200708; Abcam) was used, and GAPDH antibody (1:1000, LK9002; Tianjin Sungene Biotech Co., Ltd, Tianjin, China) was used as a loading control. The protein bands were quantified by ImageJ grayscale image peak analysis, which involved selecting the bands with the rectangle tool after the images were inverted to the 8-bit format.