Western blot analysis was performed using specific antibodies to detect the levels of COMP (ab11056, Abcam, Cambridge, UK), Bcl-2 (15071, Cell Signaling Technology, Danvers, MA, USA), Bax (2772, Cell Signaling Technology), caspase-3 (9662, Cell Signaling Technology), PI3k (4257, Cell Signaling Technology), p-PI3k p85 (ab182651, Abcam), AKT (9272, Cell Signaling Technology), p-AKT (Ser473, 4060, Cell Signaling Technology), and β-actin (Beijing Zhongshan Golden Bridge Biotechnology Co., Ltd., Beijing, China). After transfection and selection, the AKT agonist SC79 (2 µg/ml) was added to the COMP knockdown cells. Cells from each group were then collected and lysed to extract total protein after being treated in the same way for 24 h. Then 50 μg of each sample was used to determine the protein concentration by the BCA method. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis was used to separate proteins, which were then transferred to polyvinylidene fluoride membranes. The membranes were subsequently incubated overnight at 4 °C with the indicated primary antibodies, and then with the appropriate secondary antibodies at room temperature for 1 h. After exposing the membranes to detection reagent, images of immunoreactive bands were taken using a Tanon gel analyzer. All western blot analyses were performed in triplicate.

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