Journal: Frontiers in Oncology
Article Title: The Interaction of CD97/ADGRE5 With β-Catenin in Adherens Junctions Is Lost During Colorectal Carcinogenesis
Figure Lengend Snippet: CD97 binds β-catenin. (A) The N-terminal (NTF) and C-terminal fragment (CTF) of CD97 were associated in DLD1 cells (left panel) and in the colon of Tg(villin-CD97) mice (right panel). Input control and immunoprecipitates (IP), obtained either with IgG or with CD97 NTF- and CTF-specific antibodies (Abs), were analyzed with a CD97 Ab detecting the other fragment, as indicated. (B,C) CD97 and β-catenin co-immunoprecipitated in CD97-positive DLD1 cells [ (B) , left panel] and in colonic lysates of Tg(villin-CD97) mice [ (C) , left panel]. HEK293 cells, only slightly positive for CD97 [ (B) , right panel], and CD97 knock-out (Ko) mice [ (C) , right panel] served as negative controls. (D) Next to β-catenin, CD97 co-immunoprecipitated with E-cadherin (Ecad) in DLD1 cells (upper picture) and with Ecad and p120-catenin in Tg(villin-CD97) mice (lower picture). (E) upper panel: beads loaded with glutathione S-transferase (GST) or β-catenin-GST were incubated with lysates of HT1080 CD97(EGF125), CD97(EGF125/TM2) cells with a C-terminal truncated CD97 ( 15 ), and mock cells. Bound proteins were eluted and CD97 was detected with NTF or CTF CD97-specific Abs by western blotting. Pulldown could be demonstrated only with β-catenin-GST and lysates of HT1080 cells expressing full-length (TM7), but not expressing C-terminal truncated (TM2) CD97. In the lower panel the respective elution controls for β-catenin and GST are shown.
Article Snippet: Antibodies (Abs) The following Abs were used: Ecad (sc-7870, Santa Cruz, Heidelberg, Germany), glutathione S-transferase (GST) (MA4-004; Thermo Fisher Scientific, Darmstadt, Germany), α-catenin (GTX22981, GeneTex, Irvine, CA, USA), β-catenin (sc-7199; Santa Cruz; 610153; BD Transduction Laboratories, Heidelberg, Germany), p120-catenin (sc-13957, Santa Cruz), α-tubulin (T9026, Sigma-Aldrich, Munich, Germany), and ZO-1 (61-7300, Thermo Fisher).
Techniques: Mouse Assay, Immunoprecipitation, Knock-Out, Incubation, Western Blot, Expressing