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Fully confluent 10-cm dishes of HCT116 or HEK293T cells stably expressing FLAG-HA-tagged proteins were washed once with ice-cold Dulbecco’s Phosphate-Buffered Saline without calcium and magnesium, pH 7.4 (DPBS, Corning 21-031-CM), and immediately scraped into 500 μL lysis buffer [50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 0.5% (v/v) NP40 alternative (Millipore 492016), one tablet cOmplete protease inhibitor cocktail (Roche 04693116001) per 25 mL lysis buffer, one tablet PhosSTOP (Roche 04906837001) per 10 mL lysis buffer, and 1 mM DTT (Sigma-Aldrich 43816)]

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MPK3- and MPK6-Mediated ICE1 phosphorylation negatively regulates ICE1 stability and freezing tolerance in Arabidopsis