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For reducing SDS-PAGE, 2 µg of protein was mixed with 3 µL of 4× Bolt LDS Sample Buffer (Invitrogen), 1.2 µL of 10× Bolt Sample Reducing Agent (Invitrogen), and Milli-Q water to a total volume of 12 µL, and the samples were incubated for 10 min at 95 °C
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