Monoclonal antibodies to DIPA: a novel binding partner of p120-catenin isoform 1

Abstract

The coiled-coil domain-containing delta-interacting protein A (DIPA) is a transcription factor implicated in developmental regulation. DIPA is the first protein discovered to selectively interact with the p120-catenin (p120) isoform 1, an alternatively spliced form of p120 expressed preferentially in mesenchymal cells. Although a small fraction of p120 can be observed in the nucleus under certain circumstances, the vast majority of it associates with classical cadherins at adherens junctions. We observed for the first time that a discrete fraction of DIPA exists at cell-cell junctions, in addition to its predominantly nuclear localization. Thus, the p120-DIPA interaction may regulate cell signaling and/or transcriptional events, as has been described previously for β-catenin and the LEF/TCF transcription factor family. To facilitate further study of DIPA and to determine the physiological relevance of its interaction with p120, we have generated and characterized a panel of five DIPA-specific monoclonal antibodies (MAbs) that function in immunoblotting, immunoprecipitation, and immunofluorescence assays.

FIG. 1.

Alignment of human, canine, mouse, and rat DIPA proteins. Reference sequences are from the NCBI Protein Database, and ClustalW alignment was performed with MacVector software. The non-identical amino acids are shaded, and the coiled-coil 1 (42-90aa) and coiled-coil 2 (117-147aa) domains are boxed.

FIG. 2.

Reciprocal immunoprecipitations of exogenous DIPA. (A) Anti-Flag MAb was used to immunoprecipitate 3xFlag-DIPA from NP-40-detergent cell lysates of transduced MDCK cells, and immunoprecipitates were separated by SDS-PAGE and transferred to nitrocellulose. Dashed lines represent where individual strips of the nitrocellulose membrane were realigned after being probed separately. Anti-Flag and DIPA PAb were used as controls. The DIPA PAb was probed on the same strip as MAb 15F11 but detected on a separate channel using the Odyssey imaging system. (B) A portion of the same MDCK lysate (A) was immunoprecipitated with each MAb, anti-Flag, or an irrelevant IgG control. Precipitates were subjected to Western blotting as done above (A), but probed with anti-Flag MAb. MW, molecular weight marker.

FIG. 3.

Detection of endogenous DIPA by MAbs. Cell lysates were isolated from IEC6 (rat), NIH3T3 (mouse), HCA7 (human), HT29 (human), A431 (human), HepG2 (human), and three transduced MDCK (canine) cell lines with RIPA detergent. Lysates were separated by SDS-PAGE (60 μg per lane) and transferred to nitrocellulose. (A–E) Membranes were probed with MAbs 2G7, 3E3, 5G11, 8E11, and 15F11, respectively. (F) The same membrane used in E that was probed with DIPA PAb and anti-tubulin but detected on a separate channel using the Odyssey imaging system. 3x, lysate with over-expressing 3xFlag-DIPA; EV, lysate with empty vector; sh, lysate with stable expression of shRNA against canine DIPA.

FIG. 3.

Detection of endogenous DIPA by MAbs. Cell lysates were isolated from IEC6 (rat), NIH3T3 (mouse), HCA7 (human), HT29 (human), A431 (human), HepG2 (human), and three transduced MDCK (canine) cell lines with RIPA detergent. Lysates were separated by SDS-PAGE (60 μg per lane) and transferred to nitrocellulose. (A–E) Membranes were probed with MAbs 2G7, 3E3, 5G11, 8E11, and 15F11, respectively. (F) The same membrane used in E that was probed with DIPA PAb and anti-tubulin but detected on a separate channel using the Odyssey imaging system. 3x, lysate with over-expressing 3xFlag-DIPA; EV, lysate with empty vector; sh, lysate with stable expression of shRNA against canine DIPA.

FIG. 4.

Performance of anti-DIPA MAbs in immunofluorescence assays. DIPA MAbs were incubated with PFA-fixed and TritonX-100-permeabilized cells as labeled. Goat-anti-mouse Alexa Fluor 594 secondary antibodies were used to detect DIPA MAbs in all panels. No primary antibodies were used in the bottom panels (2nd Ab). Insets show magnifications to better visualize DIPA junctional staining (arrows).

FIG. 5.

Comparison of MAb 3E3 and DIPA PAb by immunofluorescence. MDCK cells stably expressing either non-silencing shRNA control (top) or canine DIPA-directed shRNA (bottom) were fixed, permeabilized, and probed with either DIPA PAb or the 3E3 MAb.

FIG. 6.

Epitope mapping of the DIPA MAbs. (A) Schematic of full-length DIPA and five overlapping fragments. The coiled-coil regions are labeled “CC1” or “CC2,” and the terminal amino acid residues are indicated next to each fragment. (B) Purified lysates from Rosetta BL-21 cells were separated by SDS-PAGE, transferred to nitrocellulose, and probed with each of the DIPA MAbs or anti-MBP as control. Lanes 1–6 were loaded with lysates containing MBP-tagged full-length, CC1, ΔC-term, CC2, ΔCC2, and C-term DIPA, respectively.

FIG. 6.

Epitope mapping of the DIPA MAbs. (A) Schematic of full-length DIPA and five overlapping fragments. The coiled-coil regions are labeled “CC1” or “CC2,” and the terminal amino acid residues are indicated next to each fragment. (B) Purified lysates from Rosetta BL-21 cells were separated by SDS-PAGE, transferred to nitrocellulose, and probed with each of the DIPA MAbs or anti-MBP as control. Lanes 1–6 were loaded with lysates containing MBP-tagged full-length, CC1, ΔC-term, CC2, ΔCC2, and C-term DIPA, respectively.