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Membranes were blocked, incubated with rabbit monoclonal anti-total Akt (C67E7) (1:1,000, Cell Signaling, #4691) or rabbit monoclonal anti-phospho S473 Akt (D9E) (1:2,000, Cell signaling, #4060) for 1 hour at room temperature, washed, and incubated with anti-Rabbit IgG, HRP Conjugate (1:2,500, Promega, W4011) secondary antibody for 1 hour at room temperature

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