Mouse monoclonal anti β catenin

All of the test compounds were synthesized at Southern Research Institute. Details of the chemical synthesis will be published elsewhere. Plasmid pGST-E-cadherin was provided by Dr. Gail Johnson (University of Rochester, Rochester, NY). The Super8XTOPFlash luciferase construct was provided by Dr. Randall T. Moon (University of Washington, Seattle, WA). The β-galactosidase-expressing vector was obtained from Promega. Rabbit monoclonal anti-axin2 (#2151) was purchased from Cell Signaling Technology. Rabbit polyclonal anti-cyclin D1 (#04-221) was from EMD Millipore. Mouse monoclonal anti-survivin (D-8) (sc-17779) was from Santa Cruz Biotechnology. Mouse monoclonal anti-β-catenin (#61054) was from BD Biosciences. Mouse monoclonal anti-actin (#A2228) was from Sigma. Peroxidase labeled anti-mouse and anti-rabbit secondary antibodies and ECL system were purchased from Amersham Life Science. The luciferase and β-galactosidase assay systems were from Promega. Tissue culture media, fetal bovine serum (FBS), and plastic-ware were obtained from Life Technologies, Inc. Proteinase inhibitor cocktail Complete™ was obtained from Boehringer Mannheim.

Whole cell extracts were prepared using Laemmli buffer (Invitrogen Life Technologies). Samples were run on a 10% sodium dodecyl sulfate (SDS)-polyacrylamide gel and transferred to a nitrocellulose membrane (Invitrogen Life Technologies). Membranes were blocked in 10% milk solution [Tris buffered saline with 0.2% Tween 20 (TBST)] for 2 h at room temperature and incubated with the indicated primary antibody overnight at 4°C. The membranes were washed three times for 10 min in TBST at room temperature and incubated for 2 h with the corresponding horseradish peroxidase (HRP)-conjugated secondary antibody (Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA). Proteins were detected using the enhanced chemiluminiscence system (GE Healthcare Life Sciences, Chalfont, UK) according to the manufacturer’s instructions. The primary antibodies used for western blot analysis were mouse monoclonal anti-TAZ (1:1,000; cat no. ab129153; Abcam, Cambridge, UK), rabbit polyclonal anti-CTGF (1:1,000; cat no. sc-25440), mouse monoclonal anti-GAPDH (1:1,000; cat no. sc-32233; Santa Cruz Biotechnology, Inc.), mouse monoclonal anti-Vimentin (1:1,000; cat no. 550513), mouse monoclonal anti-E-cadherin (1:1,000; cat no. 610182) and mouse monoclonal anti-β-catenin (1:1,000; cat no. 610153; BD Biosciences, Franklin Lakes, NJ, USA).

The mouse monoclonal anti-LRP6 antibody was from Santa Cruz (Santa Cruz, CA, USA). Rabbit polyclonal anti-p-LRP6 (Ser1490) was from Bioss (Woburn, MA, USA). Mouse monoclonal anti-β-catenin was from BD (Franklin Lakes, NJ, USA). Anti-phalloidin was from Life (Carlsbad, CA, USA). Mouse monoclonal anti-vinculin was from Boster (Wuhan, China). Mouse monoclonal anti-α-tubulin, acetyl-α-tubulin, tyrosine-α-tubulin were from Sigma-Aldrich (Saint Louis, MO, USA). Rabbit polyclonal anti-detyrosinated-α-tubulin, Mouse monoclonal anti-RhoA, Rac1and Cdc42 were purchased from Abcam (Cambridge, MA, USA). Rabbit polyclonal anti-phosphor-MAP1B (Thr1265) was from Novusbio (Littleton, CO, USA). Rabbit polyclonal anti-MACF1 was from Proteintech (Chicago, IL, USA). Rabbit monoclonal anti-phosphor-GSK3β (Ser9) and mouse monoclonal anti-GSK3β were from Cell Signaling (Beverly, MA, USA). Rabbit polyclonal anti-His, mouse monoclonal anti-His and anti-GST were from Origene (Rockville, MD, USA).

The following commercial antibodies and reagents were used: mouse monoclonal anti–α-tubulin (T9026); mouse monoclonal anti-vimentin (V6630); mouse monoclonal anti-acetylated α-tubulin (T6793); tetramethyl rhodamine isothiocyanate (TRITC)-conjugated phalloidin (P1951); 4′,6-diamidino-2-phenylindole dihydrochloride (DAPI, D9542) (Sigma-Aldrich, St. Louis, MO, USA); mouse monoclonal anti–β-catenin (#610153; BD Biosciences, Franklin Lakes, NJ, USA); mouse monoclonal anti-YAP (SC-101199) and mouse monoclonal anti-phosphotyrosine (PY99, SC-7020) (Santa Cruz, Santa Cruz, CA, USA); rabbit polyclonal anti–α-tubulin (ab18251; Abcam, Cambridge, UK); mouse monoclonal anti-nestin (#33475; Cell Signaling, Danvers, MA, USA); rabbit polyclonal anti EB3 (AB6033; Merck-Millipore, Darmstadt, Germany); AlexaFluor 488-conjugated donkey anti-mouse (A21202); AlexaFluor 488-conjugated goat anti-rabbit (A11008); AlexaFluor568-conjugated goat anti-mouse (A11031); AlexaFluor568-conjugated donkey anti-rabbit (A10042) (Thermo Fisher Scientific, Waltham, MA, USA).

The following commercial antibodies and reagents were used: mouse monoclonal anti–α-tubulin (T9026); mouse monoclonal anti-vimentin (V6630); tetramethylrhodamine isothiocyanate (TRITC)-conjugated phalloidin (P1951) (Sigma-Aldrich); mouse monoclonal anti–β-catenin (#610153; BD Biosciences); mouse monoclonal anti-YAP (SC-101199) and mouse monoclonal anti-phosphotyrosine (PY99; SC-7020) (Santa Cruz); AlexaFluor 488-conjugated donkey anti-mouse (A21202) (Invitrogen-Molecular Probes). The following small-molecule compounds were used: IB, HKL, and TDBA, dissolved in dimethylsulfoxide (DMSO) [10 (link)–12 (link)].

The following antibodies were used for immunostaining, or western blotting: mouse monoclonal anti-PLZF (Santa Cruz Biotech, Cat# sc-28319), rabbit polyclonal anti-PLZF (Santa Cruz Biotech, Cat# sc-22839), mouse monoclonal anti-γ-H2A (Millipore, Cat# 05-636), rabbit monoclonal anti-Cyclin D1 (Cell Signaling Technology, Cat# 2978), rat monoclonal anti-BrdU (Abcam Cat# ab6326), rabbit polyclonal anti-GDNF (Abcam, Cat# ab18956), rabbit polyclonal to anti-CDKN2AIP (Abcam, Cat# ab140519), rabbit polyclonal anti-MVH (Abcam, Cat# ab13840), rabbit monoclonal anti-Wilms Tumor Protein (Abcam, Cat# ab89901), mouse monoclonal anti-β-Catenin (BD Biosciences, Cat# 610154), goat Polyclonal anti-c-kit (R&D Systems, Cat# AF332-SP).

A549 and MDCK cells were cultured in glass slides. After confluency, cells were fixed with 3.7% formaldehyde and permeabilized with 0.2% Triton X-100. Cells were incubated with mouse monoclonal anti-E-cadherin (1:50, BD Biosciences, San Jose, CA, USA), mouse monoclonal anti-β-catenin (1:200, BD Biosciences, San Jose, CA, USA), mouse monoclonal anti-N-cadherin (1:50, BD Biosciences, San Jose, CA, USA) overnight at 4 °C, and they were washed three times with PBS for 15 min each. Subsequently, the cells were incubated with alexa fluor 488 goat anti-mouse IgG secondary antibodies (1:100 for E- and N-cadherin and 1:400 for β-catenin, Thermo Fisher Scientific, Waltham, MA, USA) and with 2 µg/ml of Hoechst 33342 dye for nuclear staining for 60 min at room temperature. After incubation, the samples were rinsed 3 times with PBS for 15 min each. Finally, the samples were examined under an inverted fluorescence microscope (Olympus, Tokyo, JP).