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3 protocols using multi beads shocker

1

Quantitative Western Blot Analysis of Lung Tissue

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Western blot was performed as previously described [25 (link)]. Briefly, lung tissue sample was homogenized using Multi-beads shocker® and added to the T-PER reagent (Thermo Fisher Scientific) consisting of proteinase and dephosphorylation inhibitors (Thermo Fisher Scientific). Total tissue protein purified from lungs were separated by SDS-PAGE gels and then transferred to 0.22-μm PVDF membranes (Bio-Rad). After blocking, the membranes were incubated with primary antibodies against NF-kB p65 (1:500 dilution, Abcam), IκBα (1:1000 dilution, CST), TGF-β (1:1000 dilution, Abcam), pSmad2 (1:1000 dilution, Abcam), α-Tubulin (1:1000 dilution, CST), or GAPDH (1:1000 dilution, Abcam), respectively; and followed by the appropriate horseradish peroxidase-conjugated secondary antibodies (Dako). The expression was visualized using an enhanced chemiluminescence detection kit (Thermo Scientific). Semiquantitative analysis was done using ImageQuant LAS 4000 mini (GE Healthcare Life Sciences).
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2

Quantifying Wound Angiogenesis and Inflammation

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To measure the CD31 and F4/80 protein levels, mice were sacrificed and wound tissues were harvested 7 days after treatment. Briefly, the harvested wound tissues were homogenized using Multi-beads shocker® and added to the T-PER Reagent (Thermo Fisher Scientific) consisting of proteinase and dephosphorylation inhibitors (Thermo Fisher Scientific). Total protein lysates were obtained after the elimination of debris by filtering with 0.22 μm filters (Millipore). Total protein (10 μg) was separated using a mini-protean TGX Stain-FreeTM gel (BioRad), and then transferred to PVDF membranes using the Trans-Blot® TurboTM transfer system (BioRad). After blocking with the PVDF Blocking Reagent Can Get Signal® (TOYOBO) for 1 hour at room temperature, the membranes were incubated with primary antibodies against CD31, F4/80, or tubulin (Sigma-Aldrich) at 4 °C overnight, followed by the appropriate horseradish peroxidase-conjugated secondary antibodies at room temperature for 1 hour. Expression was visualized by chemiluminescence with a digital luminescent image analyzer (LAS-4000, GE Healthcare).
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3

Inflammatory Cytokine Quantification in Lung Tissue

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To evaluate the inflammatory response, ELISA kits (R&D Systems) were used to detect the contents of transforming growth factor β1 (TGF-β1) and interleukin-1beta (IL-1β) in lung tissue lysates as previously described [25 (link)]. Briefly, the lung tissues were homogenized using Multi-beads shocker® and added to the T-PER reagent (Thermo Fisher Scientific) consisting of proteinase and dephosphorylation inhibitors (Thermo Fisher Scientific). Lung tissue lysates (100 µg protein) were added to each well and measured following the manufacturer’s instructions. The optical density was measured at 450 nm using a microplate reader (Multiskan Fc, Thermo Fisher Scientific).
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