Bca protein quantitative kit

To investigate the effect of NPWT on the activities of MMP2 and MMP9 in granulation tissue, gelatin zymography was used to detect the changes in MMP2 and MMP9 activity in granulation tissue of the eight patients with diabetic foot ulcers prior to and following NPWT for one week. The procedure for gelatin zymography is follows: The granulation tissue of eight patients with DFUs prior to and following NPWT for one week was lysed on ice and homogenate. The mixture was centrifuged at room temperature at 12,000 × g for 10 min and then, the supernatants were aspirated and the total protein was extracted. The protein content was detected by a BCA protein quantitative kit (Shanghai Biyuntian Biotechnology Co., Ltd.). Subsequently, according to the instructions of the matrix metalloproteinase (MMP2, MMP2) gelatin zymogram kit (Shanghai Xin Fan Biological Technology Co., Ltd.), the activities of MMP2 and MMP9 were determined. Briefly, 60 µg protein samples were directly loaded and separated by 10% SDS-PAGE. Following electrophoresis, the gel was washed in eluent three times, for 10 min each time and then incubated at 37°C for 6 h. After incubation, 0.05% Coomassie brilliant blue was stained for 2 h at room temperature and then decolorizing for 2 h and the gray value was analyzed using ImageJ 1.36b (National Institutes of Health).

The treated cells were collected, followed by the extraction of their complete protein. The density of protein was then analyzed by BCA protein quantitative kit (Beyotime, China). Then, 20 µg total protein was introduced into each well, segregated by 10% SDS-PAGE, then moved to a PVDF membrane by wet transfer method. After that, the layer was securely covered with 5% skimmed milk powder, with a supplement of primary antibody target gene PTEN (1:1000, ab32199, Abcam, UK) or internal reference gene β-actin (ab8226, Abcam, UK) respectively for hybridization at 4°C overnight. The gray values of each band were calculated by Image J software.

We purchased fetal bovine serum (FBS) and Dulbecco’s Modified Eagle Medium (DMEM) from GIBCO BRL (Thermo Fisher Scientific, Waltham, Massachusetts, US); Nuclear Protein Extraction Kit and IL-6 were purchased from Sigma-Aldrich (St. Louis, Missouri, US); a RNA Extraction Kit from QIAGEN (Hilden, Germany); Matrigel was purchased from BD Biosciences (Franklin Lakes, NJ, USA); an Immunohistochemistry (IHC) Kit from Thermo Fisher; and methyl thiazolyl tetrazolium (MTT), a Bicinchoninic Acid (BCA) Protein Quantitative Kit and a Dual Luciferase Reporter Gene Assay Kit from Beyotime Institute of Biotechnology (Nanjing, China). Antibodies against signal transducer and activator of transcription 1 (STAT3), STAT3 inhibitor (stattic) and β-actin were purchased from Cell Signaling Technology (CST; Danvers, MA, USA); other analytical-grade chemicals were obtained from other commercial sources.