Computational protocol: Simultaneous silencing of TGF-β1 and COX-2 reduces human skin hypertrophic scar through activation of fibroblast apoptosis

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Protocol publication

[…] Total cell lysates were prepared by solubilization in RIPA buffer containing protease and phosphatase inhibitors (Selleck Chemicals). Protein concentration was measured by the Bradford method. After 4%–20% SDS–PAGE, proteins were transferred to 0.45 um PVDF membranes (Millipore), blocked in 0.5% skim milk for 1 h at room temperature, incubated overnight with the primary antibody and secondary antibody for 1 h, with three washes of 10 min in TBS plus 0.1% Tween-20 after each incubation. The proteins recognized by the specific antibodies were visualized by chemiluminescence methods (High-sig ECL Western Blotting Substrate, Tanon, China) using peroxidase-conjugated anti-rabbit secondary antibodies as mentioned above. Densitometric quantification was performed in unsaturated images using ImageJ (NIH). [...] Mean ± SD was used for cell culture results, and mean ± SE was used for in vivo results. Student's t- test was used to determine significance between two groups. P- values < 0.05 were considered statistically significant. IBM SPSS statistics ver. 20 was used for statistical analysis. […]

Pipeline specifications

Software tools ImageJ, SPSS
Applications Miscellaneous, Microscopic phenotype analysis
Organisms Homo sapiens, Mus musculus
Diseases Skin Diseases, Wounds and Injuries