Rabbit anti yap

Formalin-fixed paraffin embedded sections were dewaxed and rehydrated. Sections were treated with 3% hydrogen peroxide in methanol for 30 min to block endogenous peroxidase activity. Antigen retrieval was performed in 10 mM sodium citrate buffer pH6 at 95°C for 40 min. To block non-specific binding sites, tissues were incubated with 1.5% normal serum (Vector Laboratories, Burlingame, CA, USA) for 30 min. Next, they were incubated with mouse anti-Argpyrimidine (mAb6B, 1:2000), rabbit anti-YAP (Santa Cruz (Dallas, TX, USA), H125, 1:100), mouse anti-Ki67 (Dako, Agilent Technologies, Santa Clara, CA, USA, 1:100), mouse anti-Glo1 (BioMac (Leipzig, Germany), 1:100) and mouse anti-vimentin (Ventana, Roche, 1:4) antibodies overnight at 4°C followed by incubation with an anti-mouse or anti-rabbit biotinylated secondary antibody (Vector Laboratories) for 30 min at room temperature (RT). Sections were then stained with avidin-biotin-peroxidase complex (Vectastain ABC Kit, Vector Laboratories) for 30 min followed by staining with 3,3’ diaminobenzidine tetrachloride (DAB). Slides were finally counterstained with hematoxylin, dehydrated and mounted with DPX (Sigma-Aldrich). Tissue sections incubated without primary antibody showed no detectable immunoreactivity.

Proteins were loaded on SDS-PAGE and electro-transferred on nitrocellulose membranes. Immunoblotting was performed according to the manufacturer’s instructions. The following primary antibodies were used: mouse-anti-Cbfβ (Santa Cruz Biotechnology Cat# sc-56751, RRID:AB_781871), rabbit-anti-MMP13 (Abcam Cat# ab39012, RRID:AB_776416), rabbit-anti-Yap (Santa Cruz Biotechnology Cat# sc-15407, RRID:AB_2273277), mouse-anti-GAPDH (Santa Cruz Biotechnology Cat# sc-365062, RRID:AB_10847862), mouse-anti-Active-β-catenin(Millipore Cat# 05–665, RRID:AB_309887), rabbit-anti-Smad3 (Cell Signaling Technology Cat# 9513, RRID:AB_2286450), and rabbit-anti-pSmad3 (Cell Signaling Technology Cat# 9520 (also 9520 S, 9520 P), RRID:AB_2193207). Secondary antibodies were goat anti-rabbit IgG-HRP (Santa Cruz Biotechnology Cat# sc-2004, RRID:AB_631746), and rabbit anti-mouse IgG-HRP (Santa Cruz Biotechnology Cat# sc-358917, RRID:AB_10989253). Quantification of Western blot area was performed by ImageJ.

Western blot was done by running 10% SDS-PAGE, following standard blotting protocol. Primary antibodies used in this study include: rabbit anti-cleaved Caspase 3 (1:1000, Cell Signaling), rabbit anti-YAP (1:1000, Santa Cruz), rabbit anti-Phospho-YAP (1:500, Cell Signaling), rabbit anti-Lats1 (1:500, Cell Signaling), rabbit anti-Phospho-Lats1 (1:500, Cell Signaling), rabbit anti-Phospho-Mst1/2 (1:500, Cell Signaling), and mouse anti-α-Tubulin (1:2000, Sigma) antibodies. Secondary antibodies are donkey anti-rabbit or mouse IgG antibodies (Amersham).