Sc 853

SW480 cells at 1 × 10⁶ cells/mL were treated with different concentrations of inhibitors for 24 h. Cells were lysed in buffer containing 50 mM Tris (pH 7.4), 150 mM NaCl, 1% Nonidet P-40, 0.5% sodium deoxycholate, 0.1% SDS, and protease inhibitors. After centrifugation at 12,000 rpm for 20 min at 4 °C, the supernatant was loaded onto an 8% SDS polyacrylamide gel for electrophoretic analysis. Separated proteins were transferred onto nitrocellulose membranes for immunoblot analysis. The antibodies against total β-catenin (610153, BD Biosciences, most of which is phosphorylated β-catenin and represents the E-cadherin bound pool), the active form of β-catenin (ABC, 05–665, EMD Millipore, dephosphorylated at positions S37 and T41 of β-catenin), cyclin D1 (sc-853, Santa Cruz Biotechnology, Inc.), c-myc (D84C12, Cell Signaling), and β-tubulin (sc-55529, Santa Cruz Biotechnology, Inc.) were incubated with the membranes overnight at 4 °C, respectively. IRDye 680LT goat antimouse IgG (827–11080, LiCOR) or IRDye 800CW goat antirabbit IgG (827–08365, LiCOR) was used as the secondary antibody. The images were detected by the Odyssey Infrared Imaging System (LiCOR). Experiments were performed in duplicate.

SW480 cells at 1 × 10⁶ cells/mL were treated with different concentrations of inhibitors for 24 h. MDA-MB-231 cells at 1 × 10⁶ cells/mL were treated with Wnt3a (100 ng/mL) for 30 min, then the inhibitors at different concentration were added and incubated for 24 h. Cells were lysed in buffer containing 50 mM Tris (pH 7.4), 150 mM NaCl, 1% Nonidet P-40, 0.5% sodium deoxycholate, 0.1% SDS, and protease inhibitors. After centrifugation at 12 000 rpm for 20 min at 4 °C, the supernatant was loaded onto an 8% SDS polyacrylamide gel for electrophoretic analysis. Separated proteins were transferred onto nitrocellulose membranes for immunoblot analysis. The antibodies against Axin2 (MA5–15015, Thermo Fisher. Immunogen: residues surrounding Pro566 of human Axin2), cyclin D1 (sc-853, Santa Cruz Biotechnology. Immunogen: human cyclin D1 residues 1−295), and β-tubulin (sc-55529, Santa Cruz Biotechnology) were incubated with the membranes overnight at 4 °C. IRDye 680LT goat antimouse IgG (827–11080, LiCOR) or IRDye 800CW goat antirabbit IgG (827–08365, LiCOR) was used as the secondary antibody. The images were detected by the Odyssey Infrared Imaging System (LiCOR). Experiments were performed in triplicate.

hPFs were from Dr. Prudence Talbot (UC Riverside) and transformed nontumorigenic human lung epithelial cells (BEAS-2B) were from ATCC (Manassas, Virginia). Normal human skin fibroblasts (HCA2) were from J. Smith (University of Texas, USA) and were immortalized by infection with an hTERT expressing retrovirus [Rubio et al. 2002 (link)]. XPA deficient patient skin fibroblasts XP12BE were from Coriell (Camden, NJ).
Antibodies used were 53BP1 (A300–272A, Bethyl), phospho-RPA32 (S4/S8: A300–245A, Bethyl), RPA32 (A300–244A, Bethyl), phospho-H2AX S139-clone JBW301 (EMD Millipore), CHK1 (2345, Cell Signaling), pCHK1-Ser317 (2344S, Cell Signaling), pATM (S1981, Ab81292, Abcam), ATM (Cell Signaling), PCNA (Santa Cruz), pBRCA1 (S1423, A300–008A, Bethyl), pATR (Thr1989, GTX128145, GeneTex), Tubulin (ab4074, Abcam) and GAPDH (MAB374, EMD Millipore) and rabbit anti-XPA (sc853, Santa Cruz).