PC12 cells were collected and freeze-thawed five times using liquid nitrogen. The cell lysates were grouped into two aliquots, with one serving as the control and the other being treated with ICA (20 μM) for 4 h at 4°C. Then, the lysates were heated at indicated temperatures (48 and 66°C, respectively) for 5 min followed by cooling at room temperature. The protein bans were detected by immunoblot [24, 26].
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