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Western blot analysis

JP Juan-Yu Piao
SK Su-Jung Kim
DK Do-Hee Kim
JP Ji Hyun Park
SP Sin-Aye Park
HH Hyeong-jun Han
HN Hye-Kyung Na
KY Kichul Yoon
HL Ha-Na Lee
NK Nayoung Kim
KH Ki Baik Hahm
YS Young-Joon Surh
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The cell lysates were prepared, and the protein concentration was measured as described previously7. The equivalent amounts of proteins (10–30 µg) were subjected to electrophoresis on 8% or 12% SDS–polyacrylamide gel and transferred to PVDF membrane. The transferred proteins were blocked in 5% fat-free dry milk in phosphate-buffered saline (PBS) containing 0.1% tween 20 (PBST) for 1 h at room temperature. Then, the membranes were incubated with primary antibodies in 3% fat-free dry milk in PBS overnight in 4 °C. Membranes were washed followed by incubation with 1:3000 dilution of respective HRP conjugated secondary antibodies for 1.5 h and again washed with PBST. Protein expressed was visualized with an ECL chemiluminescence detection kit.

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