Western Blotting and Protein Stability Assay

Western blotting was performed as previously described (Xue et al., 2022 (link); Yu et al., 2022 (link)). In brief, cell lysates were isolated on an SDS-PAGE gel and then transferred onto polyvinylidene difluoride membranes(Millipore, Bedford, MA, USA). The membranes were blocked in Tris-buffered saline tween (TBST) containing 5% fat-free milk for 1 h at room temperature, and incubated with the indicated dilutions of primary antibodies (anti-BAG3, 1:500; anti-INTS7, 1:1,000; and anti-tubulin, 1:1,000; all from Proteintech) at 4 °C overnight. The next day, the membranes were washed and treated with horseradish peroxidase (HRP)-conjugated secondary antibodies at room temperature for 1 h. Western blots were developed and visualized using the Enhanced Chemiluminescent Substrate (Thermo Scientific, Waltham, MA, USA). Tubulin was used as a loading control. For protein half-life assay, BMMSCs was incubated with 100 µg/mL cycloheximide (CHX) to block protein synthesis, and proteins were assayed after 0, 4 and 8 h.