Peroxiredoxin 1

Tomentosin was purchased from MCULE (Palo Alto, CA, USA) and dissolved in DMSO (Sigma-Aldrich, St. Louis, MO, USA). A 40 mM stock solution of tomentosin was stored at −20 °C. Antibodies for caspase-3, caspase-7, caspase-9, PARP, cleaved PARP, H2AX, γH2AX, Akt, JNK, ERK, pERK, Bcl-xl, Bcl-2, Bax, and peroxiredoxin-1 were purchased from Cell Signaling Technology (Danvers, MA, USA). Antibodies against caspase-8, pAkt (Ser473), pJNK, p38, pp38, were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Anti-rabbit immunoglobulin G (IgG) and anti-mouse IgG were purchased from Cell Signaling Technology.

The total proteins of ESCC cells and tissues were extracted with Total Protein Extraction (Sangon Biotech, China). The protein extracts were separated with SDS-PAGE after their concentrations were determined using Bradford protein assay kit (Sangon Biotech, China) and then transferred to polyvinylidine difluoride microporous (PVDF) membranes (Millipore, USA). The blotted membranes were treated with 5% (w/v) skimmed milk in TBST buffer (10 mmol/L Tris, 150 mmol/L NaCl, and 0.2% Tween 20), and incubated for 1 h at room temperature with primary antibody anti Ets2 (Santa Cruz Biotechnology, USA), p-p70S6K (Cell Signaling Technology, USA), Peroxiredoxin 1 (Cell Signaling Technology, USA) and caspase-3 (Abcam, USA), E-cadherin (Abcam, USA) and GAPDH (Cell Signaling Technology, USA). They were then washed and incubated for 1 h with a horseradish peroxidase-linked anti-rabbit secondary antibody (Sangon Biotech, China). Finally, the bands of specific proteins on the membranes were examined by Enhanced Chemiluminescence Kit (Beijing ComWin Biotech Co., Ltd, China) and densitometric analysis was performed with Image J software (NIH).