Anti wnt5a 6f2

Cells were lysed with RIPA buffer supplemented with a protease inhibitor cocktail (1:50, Roche) and phosphatase inhibitor cocktail (1:100, Thermo Fisher Scientific). After incubation on ice for 20 minutes, the lysate was centrifuged at 10,000 rpm for 15 minutes at 4°C and the supernatant was collected. Protein was quantitated by the GeneQuant 1300 (GE Healthcare) and loaded at equal amounts to each well of a 4%–20% SDS-PAGE gel. Immunoblots were performed using the following primary antibodies: anti-RUNX3 D6E2 (1:1,000; Cell Signaling Technology), anti-WNT5A 6F2 (1:1,000; LSBio), anti-GAPDH 14C10 (1:1,000; Cell Signaling Technology). Proteins were separated by SDS-PAGE and transferred to polyvinylidene difluoride membranes (Bio-Rad). After blocking in 5% milk in Tris-buffered saline with Tween (TBST) (0.1% Tween-20), the membranes were incubated with primary antibodies. Signals were detected by horseradish peroxidase–conjugated secondary anti-rabbit (1:10,000, GE Healthcare) or mouse antibody (1:10,000, GE Healthcare) and visualized with Image Quant LAS 500 (GE Healthcare).

Paraffin section slides were deparaffinized, described as above. In brief, sections were pretreated by autoclave at 121°C for 20 minutes in antigen retrieval solution (DAKO) to retrieve antigenicity. Sections were blocked by incubation in 5% skim milk or protein block serum-free (DAKO). Primary antibodies specific for anti-RUNX3 D6E2 (1:250; Cell Signaling Technology) and anti-WNT5A 6F2 (1:400; LSBio) were applied to the slides and incubated at 4°C overnight. The samples were treated with conjugated secondary antibody (Invitrogen). Nuclei were stained with 4′,6-diamidino-2-phenylindole (Sigma). Cells were then analyzed by fluorescence microscopy (Zeiss). RUNX3-expressing lymphocytes were distinguished from epithelial cells by E-cadherin. E-cadherin (1:200; BD Pharmingen) or Ki67 (1:250; Invitrogen) positive cells were screened when we counted RUNX3 positive or WNT5A positive cells in gastric cancer specimens.