β catenin antibody

Cinobufagin (CBG) was purchased from Pusi Biotechnology Co. Ltd, and the purity of CBG was 99.21%. For each experiment, CBG was freshly prepared by dissolving in DMSO (Sigma, USA). Bleomycin (BLM) was acquired from Nippon Kayaku (Tokyo, Japan). TRIzol reagent was from Ambion Life Technology. DEPC TREATED H₂O, RNASE AWAY H₂O and SYBR Green Real-time PCR Master Mix were from Life Technologies. M-MLV Reverse Transcriptase was purchased from Promega, and PCR Buffer without MgCl₂ and MgCl₂ Stock Solution were acquired from Roche. In addition, Recombinant Ribonuclease Inhibitor, dATP, dTTP, dCTP, and dGTP were acquired from Takara. RIPA lysis buffer (middle) and the BCA kit were purchased from Beyotime Biotechnology. The primary antibodies described in the study include: anti-E-cadherin, anti-Vimentin, anti-fibronectin, and anti-collagen I (Affinity Biosciences, OH, USA) and anti-GAPDH, Smad3, P-Smad3, ERK, P-ERK, JNK, P-JNK, P38, P-P38 antibody (Cell Signaling Technology, Boston, United States); α-SMA and β-catenin antibody were from Santa Cruz Biotechnology (China) and Protein-Tech (China), respectively. The secondary antibodies anti-rabbit IgG (H+L) and anti-mouse IgG (H+L) were from Applygen (Beijing). Polyethylenimine, Linear purchased from Tianjin Hao Trading. β-catenin plasmid was purchased from MiaoLingPlasmid. ELISA kits were purchased from Beijing Suobao Technology.

The mouse prostate tissues were fixed in 10% formaldehyde and embedded in paraffin wax. Five-micrometer sections were cut and stained with H&E for pathological evaluation. Sections were also stained with antibodies specific for: (1) E-cadherin (Santa Cruz); (2) p63 (Thermo Scientific); (3) Ki67 (Thermo Scientific); (4) β-catenin antibody (AbCAM); (5) AR (Santa Cruz); (6) pSTAT3 (Cell signaling); (7) macrophage (F4/80 or Mac-2; eBioscience); (8) B cells (B220, eBioscience); and (9) anti-CD3 (Thermo Scientific). The staining procedure has been previously described [14 (link)]. Briefly, sections were deparaffinized and incubated for 10 min in 10 mM citrate buffer (pH 6.0) at 95 °C for antigen retrieval. Endogenous peroxidase activity was quenched with 3% hydrogen peroxide in methanol. After quenching endogenous peroxidase activity and blocking nonspecific binding, slides were incubated with specific primary antibody overnight at 4 °C followed by subsequent incubation with the appropriate biotinylated secondary antibody provided with Vectastain Elite ABC Kit. Color was developed with DAB as the perioxidase substract. All slides were counterstained with hematoxylin and mounted with Permount. Ten randomly selected fields of IHC-stained sections of the prostates from individual mice were counted for the positively stained cells and used for statistical analysis.