Figure 1

APC-NT mAbs recognize endogenous and recombinant full-length and truncated APC proteins in solution. A) Schematic diagram of structures of recombinant APC proteins. The APC-NT antigen (APC residues 1–61 with an N-terminal FLAG-tag) was expressed and purified from E.coli and used to generate the APC-NT mAbs. Full-length (fl-APC) and truncated recombinant APCs APC(1–1638) and APC(1–1311) were expressed in Sf9 cells with N-terminal HIS and C-terminal EE tags. The protein domains of APC are indicated: Oligomerisation, Armadillo repeats, 15 aa repeats, 20 aa repeats, SAMP motifs, basic rich domain, PDZ domain. Note: the EE-epitope tag was not used for the final affinity purification. B) Immunoprecipitation of endogenous full-length and truncated APC. APC-NT mAb immunoprecipitates from MDCK cells (1 mg protein, left panel) and SW480 colorectal carcinoma cells (1 mg protein, right panel) were immunoblotted (IB) with anti-APC H290. C) Biosensor analysis shows overlapping and non-overlapping epitopes for the APC-NT mAbs. I) Representative sensorgram showing sequential injection of APC-NT mAbs and binding to APC-NT antigen. II) Stack graph showing increases in binding (RU) to APC-NT antigen upon sequential injections with different APC-NT mAb clones. Four combinations of APC-NT mAb clones injected sequentially are represented by shaded areas (A-D). Column A corresponds to the sensorgram in I). 2E7 and 8D9 have different epitopes whereas 6D12 and 6G6 share overlapping epitopes. D) Immunoprecipitation of recombinant APC proteins expressed in Sf9 cells. APC-NT mAb (2 μg) immunoprecipitates from Sf9 cell lysates (2×10⁷ cells) expressing fl-APC (left), APC(1–1638) (middle) or APC(1–1311) (right) were resolved by SDS-PAGE and immunoprecipitated proteins were visualised with Coomassie blue. Note protein immunoprecipitated by 6D12 and 6G6 migrate slightly differently. The relative positions of the molecular markers are indicated.