β catenin

The primary antibodies for Bak, pFAK, Bad, Bax, Bcl2, and PARP were purchased from Cell Signaling (Danvers, MA, USA). Rac1 and Cdc42 antibodies were obtained from Millipore (Billerica, MA, USA). The FAK antibody was obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA), p53 and NF-kB antibodies were purchased from Neomarkers (Fremont, CA, USA), the vimentin and E-cadherin antibody were obtained from BD Biosciences (CA, USA), and β-catenin, Smad 2, and 3 antibodies were obtained from Genetex (Irvine, CA, USA). The antibody for β-actin was obtained from Sigma (USA), the anti-mouse and anti-rabbit IgG horseradish peroxidase-conjugated secondary antibodies were purchased from GE Healthcare (UK). PF-431396 was purchased from Sigma Aldrich. Synthetic antroquinonol [24 (link)] was obtained from Dr. Chin Po Chen (Department of Chemistry, NDHU, Hualien, Taiwan).

Total protein was extracted from cells using whole-cell extract (WCE) lysis buffer. Lysates were vibrated for 30 min and centrifuged at 13,200 rpm for 20 min at 4°C. Western blotting was performed as outlined in our previous report [24 (link)], and the fractionation protocol used has been described previously [49 (link)]. Primary antibodies include RPIA (Cat# ab67080; Abcam, Cambridge, United Kingdom), β-actin (Cat# GTX109639; GeneTex, Inc, Irvine, CA), β-catenin (Cat# GTX61089, GeneTex; Cat# ab22656, Abcam), β-catenin (phospho Ser33/Ser37) (Cat# GTX11350 GeneTex), APC (Cat# GTX61328, GeneTex), Ubiquitin (Cat# 3936; Cell Signaling Technology, Danvers, MA), K48-linkage specific polyUbiquitin (Cat# 4289, Cell Signaling Tecnology), β-Tubulin (Cat# ab52866, Abcam), and Lamin A/C (Cat# ab108922, Abcam). For IP, 100 μg of protein lysate was incubated with primary antibody overnight and subsequently incubated with protein A/G-Sepharose beads for 1.5 h. The interaction results were assessed with western blotting.

Most chemicals used and Protein A-agarose were purchased from Sigma-Aldrich (St. Louis, MO, USA). The antibody for CCT-β was purchased from Abcam (Cambridge, MA, USA) for the co-IP assay. For western blots, the specific antibodies for GAPDH (100118), CCT-α (16399), CCT-β (112283), CCT-γ (33073), CCT-δ (33536), CCT-ε (110167), CCT-ζ (105148), CCT-η (101347), CCT-θ (115466), XIAP (111202), β-catenin (101435), P-β-catenin (S675) (132611), P-β-catenin (Ser33/Ser37/Thr41) (132605), Slug (121924), Snail (100754), Twist (127310), E-cadherin (100443), MUC1 (100459), N-cadherin (112734), MMP2 (104577), MMP9 (100458), AKT (121973), P-AKT (128414), GSK-3β (111192), P-GSK-3β (50090), Survivin (100052), NF-kB p65 (102090), fibronectin (112794), β1 (112971), Src (134412), P-Src (54701), FAK (50489), and P-FAK (129840) were from GeneTex (Irvine, CA, USA). The catalog numbers are in parentheses.

Whole-cell extracts were prepared by scraping in 0.2–0.3 mL of RIPA lysis buffer as described earlier (Tsai et al. 2015 (link)). Nuclear and cytoplasmic fractions were harvested by using the Nuclear Extract Kit and following the manufacturer’s instructions (Active Motif, Carlsbad, CA). Protein concentrations were determined by Bradford method (BioRad, Hercules, CA). Equal amounts of protein from each treatment were diluted with loading buffer, boiled, and separated on 10 or 15 % gels by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Then, the separated proteins were transferred to polyvinylidene difluoride (PVDF) membranes. After a short blocking with non-fat milk, the membranes were probed with appropriate primary antibodies, including AHR, ARNT (Santa Cruz), vimentin, β-catenin, N-cadherin, E-cadherin, claudin-1 (Genetex), occludin (Proteintech), ZO-1 (Invitrogen), lamin A/C, GSK3β phospho-Ser-9 and phospho-Tyr-216, GSK3β (Epitomics), and β-actin (Sigma), followed by an incubation with horseradish peroxidase-conjugated secondary antibodies. Immunocomplexes were visualized using the enhanced chemiluminescence (ECL) detection reagent (Millipore). Band intensities were determined by densitometry analysis using Gel-Pro Analyzer software (Media Cybernetics, Warrendale, PA).