Imaging densitometer

After cells or tissues were lysed, we used a BCA Protein Assay Kit (Boster Bio, USA) to determine the protein concentration. Total protein was separated by SDS-PAGE. We used a constant current to transfer the separated protein to a PVDF membrane. 5% skim milk was applied to block non-specific reaction sites for 1-2 h. Dilute the primary antibody at the concentration recommended by the instructions, immerse the membrane in this liquid, and place at 4° C for at least 8 hours. The primary antibodies: anti-GAPDH (Proteintech, Wuhan, China), anti-p-ERK (CST), anti-ERK (CST), anti-NOX4 (Abcam, Cambridge, UK), anti-p-AMPK (Abcam), anti-AMPK (Abcam). Horseradish peroxidase-conjugated secondary antibodies were diluted at the concentrations recommended by the instructions, and the membranes were incubated for 2h at room temperature. Enhanced chemiluminescence reagent was used to visualize the protein bands in an imaging densitometer (GE, USA).

The protein expression of Hsp27 was examined. In order to examine Hsp27 expression, we conducted the same experiment using another 6 hairless rats according to the design protocol (3 rats in each group). We obtained skin samples from the PI wounds by surgery biopsy from different rats at 6, 12, and 24 h after decompression for protein analysis. The samples were snap frozen immediately following resection. The tissue was homogenized in ice-cold RIPA, and then centrifuged at 12,000× g for 10 min at 4 °C. The supernatant containing the total cell protein was extracted for protein concentration assay. Samples were separated on 10% sodium dodecyl sulfate-polyacrylamide gels, and then transferred onto a nitrocellulose membrane. Each well was loaded with approximately 30 μg of soluble protein. Placental tissue, which expresses Hsp27, was used as a reference sample and positive control on each gel. The primary antibody (HSP 27, Santa Cruz) was used as 1:1000. The second antibody was horseradish peroxidase-conjugated donkey anti-mouse secondary antibody (Jackson, 1:5000). Proteins were visualized using the ECL detection system (Thermo Fisher Scientific). Bands were scanned using a GE imaging densitometer. Band densities were expressed relative to the density of the internal placental control sample included on every well by using the ImageJ software.