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devilpanda金虫 (小有名气)
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Whole-cell extracts were prepared by lysing the cells with RIPA buffer containing 150 mM NaCl, 50 mM Tris HCl (pH 8), 1% NP-40, 0.5% sodium deoxycholate, 0.1% SDS, and protease inhibitor and phosphatase inhibitor cocktails (Sigma- Aldrich). The cell extracts were separated by 10% SDS-PAGE and transferred onto a polyvinylidene fluoride membrane (Millipore, Billerica, MA, USA). Samples were incubated in blocking buffer (0.1% Tween 20 and 5% nonfat milk powder in Tris-buffered saline [TBS]) for 1 h at room temperature. Afterward, the membrane was incubated with primary antibody in blocking buffer overnight at 4 °C before being washed twice with TBST (0.1% Tween in TBS) and incubated with the appropriate secondary antibody in blocking buffer for 1 h at room temperature. The blot was developed using ECL Western blotting substrate (Millipore) and analyzed using a luminescent image analyzer (LAS-4000 mini; FujiFilm, Tokyo, Japan). The primary antibodies were used at the following dilutions: rat anti-ABCG2, 1:100 (Abcam, Cambridge, UK); rabbit anti-PARP, 1:1000 (Cell Signaling Technology, Beverly, MA, USA); rabbit anti-caspase 7, 1:1000 (Cell Signaling Technology); rabbit anti-Akt and phosphate Akt-ser 473, 1:1000 (Cell Signaling Technology); and mouse anti-β-actin, 1:10000 (Sigma-Aldrich)谁能帮我翻译下啊~~  |
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铁杆木虫 (著名写手)
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