Anti p p65

Cocultured BMSCs were harvested regularly for WB experiments, with total protein extracted using a RIPA lysis buffer (Beyotime). Protein concentrations were determined using a bicinchoninic acid assay kit (Solarbio, Beijing, China). Subsequently, 20 µg of protein from each sample was loaded onto a 10% Sodium sodecyl sulfate polyacrylamide gel electrophoresis gel (New Cell & Molecular, Suzhou, China) and subjected to electrophoresis. Proteins were transferred to membranes, which were then blocked. Membranes were incubated with the primary antibodies anti‐TLR4, anti‐P65, anti‐p‐P65, anti‐STAT3, anti‐p‐STAT3, anti‐ERK, anti‐p‐ERK, and Gapdh (all sourced from Abcam). After thorough washing with Tris‐buffered saline‐Tween (Biosharp), colorimetric detection was carried out using horseradish peroxidase‐conjugated secondary antibodies. Protein bands were visualized using an imaging system (Bio‐Rad). Quantitative analysis of the proteins was performed using ImageJ.

Western blotting was used to determine the effect of SH on the expressions of apoptotic proteins in HaCaT cells [33 ,34 (link)]. The HaCaT cells were treated with SH and SH-NPs (1 μM and 2 μM) for 4 h. Subsequently, the cells were exposed to TNF-α (10 ng/ml) for 24 h. Then, total protein extraction was done by lysing the HaCaT cells with RIPA buffer at 4 °C for 25 min. The lysate was rinsed once using PBS, and with RIPA buffer for 30 min. The concentrations of total protein samples obtained after centrifugation of the lysate were measured using BCA kit. Then, equal amounts of protein samples were subjected to 10 % SDS-PAGE, followed by transfer onto PVDF membranes (Millipore, USA) which were thereafter blocked in 5 % defatted milk. The PVDF membranes were subjected to overnight incubation with primary antibodies for anti-p65 (1:1000) and anti-p-p65 (1:1000) at 4 °C (Abcam, USA), and subsequently probed using horse radish peroxidase-labeled 2^o immunoglobulins (diluted 1:8000) for 2 h at 25 °C. Blot detection was done using enhanced chemiluminescence (Thermo Fisher Scientific, USA). The internal reference was GAPDH. All experiments were independently repeated three times.