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The mixture was vortexed and centrifuged (5 min, 16,000 g) several times during 1 h

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Chen JC, Chiu MH, Nie RL, Cordell GA, Qiu SX (2005) Cucurbitacins and cucurbitane glycosides: structures and biological activities

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Immunoblots were always analyzed first for phosphorylated forms of proteins, stripped with Restore PLUS Western Blot Stripping Buffer (Thermo Fisher Scientific), and re-probed with the corresponding antibodies for total proteins