Mouse anti β catenin antibody

Cell lysates were prepared with RIPA buffer (Beyotime). Protein concentration was determined using a BCA Protein Assay kit (Pierce). Samples were separated by SDS‐PAGE, blotted onto polyvinylidene fluoride membranes, and probed with primary antibodies, followed by horseradish peroxidase–conjugated goat antirabbit IgG or goat antimouse IgG (Boster Bio Tec). β‐actin was used as a loading control. The primary antibodies included rabbit antihuman EVL (1:50; Santa Cruz Biotechnology), mouse antihuman endoglin (1:800; BD Biosciences), rabbit anti–phosphorylated Smad1/5 (1:1000; Cell Signaling), rabbit anti–phosphorylated Smad2/3 (1:500; Santa Cruz Biotechnology), rabbit anti–phosphorylated Akt (Ser 473, 1:800; Cell Signaling), rabbit anti‐Akt (1:800; Cell Signaling), rabbit anti–phosphorylated ERK (1:1000; Cell Signaling), rabbit anti‐ERK (1:1000; Cell Signaling), mouse anti‐β‐actin (1:1000; Sigma‐Aldrich), mouse anti‐CD31 antibody (1:1000; Abcam), rabbit anti–α‐smooth muscle actin antibody (1:200; Abcam), mouse anti–β‐catenin antibody (1:1000; Millipore), rabbit anti‐vimentin antibody (1:1000; Abcam). Membranes were developed using an enhanced chemoluminescence system (Clinx Science Instruments).

Cancer cells were grown on collagen-coated glass cover slips and fixed for 30 minutes in 4% paraformaldehyde/1% Triton X-100. Samples were washed and incubated with 0.3% hydrogen peroxide for 30 minutes and washed in PBS. Cells were blocked with 1% BSA (Vector Laboratories), PBS washed and incubated for 30 minutes with a mouse anti-β-catenin antibody (Millipore, Billerica, MA) at a 1:500 dilution. Slides were washed with PBS and incubated with Alexa 488 goat anti-rabbit (1:400 dilution) (Invitrogen) for 30 minutes. Cells were washed with PBS/0.1% Triton X-100. Samples were nuclear counterstained with 300 nM 4′,6-diamidino-2-phenylindole (DAPI) for 5 minutes.