Anti wnt1

The relative protein abundances in kidneys and HK-2 cells were analyzed using western blotting, as described previously (Xu et al., 2012 (link)). The following primary antibodies were used: anti-α-SMA (1:1,000, Sigma), anti-E-cadherin (1:1,000, Cell Signaling Technology), anti-vimentin (1:1,000, Santa Cruz Biotechnology), anti-collagen I (1:1,000, Abcam), anti-collagen IV (1:1,000, Abcam), anti-Wnt1 (1:100, Abcam), anti-Wnt4 (1:150, Santa Cruz Biotechnology), anti-Wnt3 (1:500, Abcam), anti-Wnt2b (1:700, Abcam), anti-Wnt7a (1:300, Abcam), anti-β-catenin (1:1,000, Cell Signaling Technology), anti-Snail 1 (1:1,000, Santa Cruz Biotechnology), anti-PAI-1 (1:1,000, Santa Cruz Biotechnology), and anti-NF-κB p65 (1:1,000, Cell Signal). Horseradish peroxidase-conjugated secondary antibodies (anti-rabbit or anti-mouse or anti-goat IgG, 1:5,000, Jackson ImmunoResearch) were used according to the manufacturer’s instructions. Image analysis software (Image J, National Institutes of Health) was used to determine the gray value of all bands.

Changes in renal morphology were examined in paraffin-embedded tissue sections (4 μm) stained with hematoxylin. Immunohistochemical staining was performed as previously described (Xu et al., 2012 (link)). The following primary antibodies were used: anti-Wnt1 (1:100, Abcam), anti-Wnt4 (1:150, Santa Cruz Biotechnology), anti-c-caspase3 (1:500, Cell Signaling Technology), anti-F4/80 (1:200, Abcam). The secondary antibody was horseradish peroxidase-conjugated anti-mouse IgG (1:1,000; Jackson ImmnoResearch), which was used according to the manufacturer’s instructions. The peroxidase was visualized with diaminobenzidine. To determine the number of F4/80-positive cells, 8–10 fields for each mouse were examined under a microscope (magnification, 200×), and the average number of positive cells was calculated.

The use of fresh breast tumor specimens was approved by the research ethic committees at the Korea National Cancer Center (NCC). Informed consent was obtained from all patients. All experiments with human specimens were performed in accordance with relevant guidelines and regulations of NCC. Samples were fixed with 4% paraformaldehyde for fluorescent staining. Samples were permeabilized with 0.3 M glycine and 0.3% Triton X-100, and nonspecific binding was blocked with 2% normal swine serum (DAKO, Glostrup, Denmark). Staining was performed as described previously56 (link), using the primary anti-Wnt1 (Abcam), anti-Phalloidin (Cytoskeleton Inc.), anti-ALDH1 (Abcam), anti-TCF41 (Abcam), anti-PCNA (Abcam), and anti-LEF1 (Cell Signaling Technology). Samples were examined by fluorescence microscopy (Zeiss LSM 510 Meta). The calculation of expression was based on green fluorescence area and density divided by cell number, as determined from the number of DAPI-stained nuclei, in three randomly selected fields for each specimen from a total of three independent experiments. For quantitation, an arbitrary threshold was set to distinguish specific from background staining, and this same threshold setting was applied to all the samples analyzed.