Proteins were extracted from the cells as previously described (7). Briefly, the hPDLSCs were rinsed in PBS, then lysed on ice in RIPA buffer (Beijing Solarbio Science & Technology Co., Ltd.) comprising 1% phosphatase inhibitor (Boster Biological Technology Co., Ltd.) plus 1% PMSF (Beijing Solarbio Science & Technology Co., Ltd.). Following ultrasonic cracking and centrifugation (12,000 × g; 20 min; 4°C), the concentration of proteins was determine using the BCA assay kit (Beijing Solarbio Science & Technology Co., Ltd.). Subsequently, the proteins from different groups were resolved on 10% SDS-PAGE and transferred onto a PVDF membrane. A total of 20 μg proteins from different groups were loaded on 10% SDS-PAGE and separated through electrophoresis. Subsequently, the separated proteins were eletro-blotted onto a PVDF membrane. Blocking was performed using 5% non-fat dry milk at 22°C for 1 h, then probed with rabbit anti-human GAPDH polyclonal (1:20,000, cat. no. 10494-1-AP; ProteinTech Group, Inc.), rabbit anti-human RUNX2 monoclonal (1:1,000, cat. no. ab23981; Abcam), rabbit anti human-COL1 mono- clonal (1:1,000, cat. no. #84336; Cell Signaling Technology, Inc.), rabbit anti human-ALP monoclonal (1:5,000, cat. no. ab108337; Abcam), rabbit anti human-NLRP3 polyclonal (1:1,000, cat. no. WL02635; Wanlei Biotech Co., Ltd.), rabbit anti human-procaspase-1 polyclonal (1:500, cat. no. WL02996; Wanlei Biotech Co., Ltd., China), rabbit anti human-caspase-1 polyclonal (1:500, cat. no. WL03450; Wanlei Biotech Co., Ltd.), rabbit anti human-phosphorylated (p-)p65 polyclonal (1:500, cat. no. WL02169; Wanlei Biotech Co., Ltd.), rabbit anti human-p-IκBα polyclonal (1:500, cat. no. WL02495; Wanlei Biotech Co., Ltd.), rabbit anti human-IκBα polyclonal (1:500, cat. no. WL01936; Wanlei Biotech Co., Ltd.), rabbit anti human-p65 monoclonal (1:1,000, cat. no. #59674; Cell Signaling Technology, Inc.) at 4°C for 24 h. This was followed by incubation with a horseradish peroxidase-labeled goat anti-rabbit IgG secondary antibody (1:20,000, cat. no. 7074S; Santa Cruz Biotechnology, Inc.) at room temperature for 1 h. Protein detection was performed using a chemiluminescent HRP (EMD Millipore) and the expression levels of target proteins were analyzed using ImageJ 1.47V software (National Institutes of Health) and normalized to GAPDH expression.

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