Western blotting of endogenous APC and GFP-APC constructs expressed in COS-7 cells. Panel A: Western blot of cell extracts from COS-7 (lane 1), HeLa (lane 2), MDCK (lane 3), NRK-52E (lane 4) and Caco-2 (lane 5), 10 μg total protein was loaded for each extract and the membrane probed with the ALI 12-28 antibody specific for the APC N-terminus. Full-length APC (310 kDa) can be detected in all cell lines except CaCo-2 cells that possess a much smaller truncated APC protein (not shown). Panel B: Western blot of COS-7 cell extracts transfected with plasmids directing the expression of GFP-APC (lane 1), GFP-APC-ΔN (lane 2), GFP-APC-ΔN+ΔC (lane 3), and untransfected COS-7 cells (lane 4). Prior to western blotting extracts were immunoprecipitated with a polyclonal anti-GFP antibody. The immunoprecipitation from each extract was loaded onto a gradient gel, subjected to SDS-PAGE, transferred to nitrocellulose and the membrane probed with a polyclonal anti-GFP antibody. Panel C: Western blot of the COS-7 cell extract transfected with the plasmid directing the expression of GFP-APC-N construct (lane 1) or a non-transfected COS-7 cell extract (lane 2). 20 μg of each protein extract was loaded onto a 10% non-gradient gel and the membrane probed with a polyclonal anti-GFP antibody.