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Phosphopeptides were eluted in three rounds with 80 l 1% ammonia for 20 min, transfered the supernatant to a clean plate, and acidified with TFA

[DOI] [PMC free article] [PubMed] [Google Scholar] 76.Minokoshi Y, Alquier T, Furukawa N, Kim YB, Lee A, Xue B, Mu J, Foufelle F, Ferre P, Birnbaum MJ, Stuck BJ, Kahn BB

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Wash with PBS one more time and add 500 l of lysis buffer (0.1% Triton-X and 0.6% sulfosalicylic acid in 0.1 M potassium phosphate buffer with 5 mM EDTA at pH 7.5) on ice