P erk1 2 thr202 thr204

The protein level in the renal tissue was estimated by means of Western Blotting. Basing on the immunohistochemistry results, we analyzed the presence of p-ERK1/2 (Thr202/Thr204; #9101, 1:1000, Cell Signaling Technology) and p-mTOR (Ser2448; #2971, 1:1000, Cell Signaling Technology) to verify the activity of these proteins in the kidneys. To reveal other possible mechanisms implicated in the HFKD action, we also examined the amounts of p53 (MAB1355, 1:500, R&D), nuclear factor erythroid 2-related factor 2 (Nrf2; MAB3925, 1:1000, R&D) and 8-oxoguanine glycosylase α (NB100-106, 1:1000, Novus Biological). The results were normalized to the β-actin reference protein (#4970, 1:1000, Cell Signaling Technology) (see also Supplementary Information).

Five micrometer paraffin sections were deparaffinized with a xylene and alcohol series, treated with Target Retrieval Solution pH 6.1 (Dako, Carpinteria, CA, USA), blocked with 3% H₂O₂ in methanol, and then placed in a 5% normal goat serum in 0.1% Triton X in TBS. The sections were incubated overnight with primary antibodies at 4 °C, were washed, and then incubated (2h) with a secondary antibody conjugated with horseradish peroxidase (HRP). AEC (3-amino-9-ethylcarbazole, Envision + System Dako) was then applied to generate a color reaction. The slides were then counterstained with hematoxylin. The primary antibodies used for labeling were: p-ERK1/2 Thr202/Thr204 (#9101, 1:100, Cell Signaling Technology); and p-mTOR Ser2448 (#2971, 1:100, Cell Signaling Technology). A visual qualitative observation (using BX51 microscope with U-TV-1X-2 camera and CellˆB image acquisition software, all from Olympus, Tokyo, Japan) was used to determine the signal intensity in the tumor tissue relative to the normal tissue.