As described, alveolar bone was ground to a powder and RIPA lysis buffer (Lot 02408/60412, CwBio Biotechnology Co., Ltd. China) used to extract proteins. Protein concentrations were determined using a bicinchoninic acid protein assay detection kit (P0012S, BeyoTime Biotechnology, China). Samples were mixed with a 1/4 volume of 5× sodium dodecyl sulfate loading buffer and heated to 95°C for 5 min. Proteins then underwent sodium dodecyl sulfate-polyacrylamide gel electrophoresis, were transferred to polyvinylidene fluoride membranes, and blocked in 5% BSA in Tris buffered saline with Tween-20 (TBST) (5% BSA-TBST) at room temperature for 1 h. Membranes were incubated overnight at 4°C with the following: (Tonetti et al., 2018 (link)) anti-p-STAT3 (1:1000; ab76315, Abcam), (Cecoro et al., 2020 (link)) anti-p-STAT5 antibodies (1:1000, AF3304, Affinity Biosciences), (Barutta et al., 2022 (link)) anti- STAT3 (1:1000; ab68153, Abcam), (Zheng et al., 2021 (link)) anti- STAT5 (1:1000; ab32043, Abcam) and followed by incubation with a horseradish peroxidase-conjugated goat anti-rabbit IgG antibody (1:1000, ab6721, Abcam) for 1 h. Immunoreactive bands were detected using enhanced chemiluminescence reagent (B500024, Proteintech, United States) and a gel imaging system (Amersham Imager 600; General Electric Company, United States) to capture images. Image-Pro Plus 6.2 software (Media Cybernetics) was used to analyze and quantify grayscale values normalized to GAPDH levels. Experiments were repeated at least three times.
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