Axin2

6-well plates were incubated with 1ml poly-D-lysine solution (Sigma, P6407, 10 μg/mL) for 30 minutes at room temperature after which time the poly-lysine solution was removed and the wells washed twice with 2 mL of distilled water. To the wells were added HCT-116 cells, (ATCC, CCL-247), maintained in McCoy’s 5A medium (Invitrogen, 16600-082) supplemented with 10% FBS (Atlanta Biologicals, S11050), and Penicillin-Streptomycin (100 U/mL) (Invitrogen, 15140122), or SW480 cells (ATCC, CCL-228), maintained in MEM growth medium (Sigma, M4655) supplemented with 10% FBS and 100 U per ml penicillin and streptomycin. The cells were allowed to grow for 48 hours at 37°C, 5% CO2 in an incubator. The cell confluency was approximately 60–80%. The media was then replaced with fresh media containing the indicated compounds in DMSO or DMSO. The final DMSO concentration was 0.1%. The cells were incubated for 18 hours. The media was removed and the cells washed 2 times with PBS, and the cytosolic fractions isolated using hypotonic buffer as described previously (Chen M, et. al. Biochemistry (2009) 48(43): 10267–10274). Cytosolic fractions were analyzed by Western blot using antibodies to β-catenin (Santa Cruz Biotechnology, SC-7963) or Axin2 (Santa Cruz Biotechnology, SC-20784). As a loading control, β-actin was analyzed with antibodies to β-actin (C-4, Santa Cruz Biotechnology, SC-47778).