Western Blot Analysis of S100A10 Protein

This part refers to our previous experimental methods20 (link). Briefly, Total cellular proteins were lysed with a radio-immunoprecipitation assay (RIPA). The concentrations of the proteins were determined using the bicinchoninic acid (BCA) assay method and equal concentrations of the proteins were loaded on SDS-PAGE gel (10%). Thereafter, the samples were transferred onto polyvinylidene difluoride (PVDF) membranes and blocked with 5% skimmed milk solution for 1 hour. Next, membrane was incubated with S100A10 rabbit monoclonal antibody (1:1000; Cat. no. 0041696; Proteintech, Wuhan, China) or GAPDH rabbit polyclonal antibody (1:5000; Cat. no. 10494-1-AP; Proteintech, Wuhan, China) at 4°C overnight. After washing with PBST for 4 times, the membranes were incubated with horseradish peroxidase-linked secondary biotinylated antibodies for 1.5h at room temperature. Following washing with PBST for 4 times, the immunoreactive bands were observed using the enhanced chemiluminescence (ECL) reagent (Vazyme, Nanjing, China), and the images were recorded in the Gel Imaging System. The relative S100A10 expression levels were calculated by the Gel-Pro-Analyzer (Tanon, Shanghai, China).