Anti laminin γ2

Formalin‐fixed and paraffin‐embedded tissue sections were stained with anti‐Daple (1:100; IBL, Gumma, Japan), anti‐Wnt5a/b (1:50; Cell Signaling Technology, Danvers, MA, USA), anti‐laminin γ2 (1:200; Millipore, Bedford, MA, USA) and anti‐β‐catenin (1:1000; BD Transduction Laboratories, San Jose, CA, USA) antibodies. The sections were pretreated by boiling in citrate buffer (pH 7.0) for Daple, Wnt5a/b and β‐catenin staining or by incubation with proteinase K for laminin γ2 staining. After blocking with Protein Block Serum Free (Dako, Glosturp, Denmark), the sections were incubated with primary antibodies overnight at 4°C, then with secondary antibodies (Envision+, Dako). Reaction products were visualized using diaminobenzidine (Dako).

Cells were lysed in SDS sample buffer. GTP loading of Rac was determined by pull‐down assay using GST‐PAK‐PBD (Cytoskeleton, Denver, CO, USA). Samples were separated by SDS‐PAGE, and proteins were transferred to PVDF membranes (Millipore). The membranes were blocked in 4% skimmed milk, and probed with anti‐Daple (1:500), anti‐laminin γ2 (1:1000), anti‐Wnt5a/b (1:1000), anti‐Rac1 (1:1000; Millipore) and anti‐phospho JNK and anti‐JNK (1:100; Cell Signaling Technology) antibodies. After incubation with HRP‐conjugated secondary antibodies (Dako), immunoreactivity was detected with an enhanced chemiluminescence system (Amersham Biosciences, Piscataway, NJ, USA).