Association of endogenous cadherins with CDO and BOC. (A and B) Association of CDO, cadherin, and β-catenin in C2C12 cells. Lysates from proliferating, low-density C2C12 cells cultured in growth medium (G) or confluent cultures in differentiation medium [2% horse serum (HS)] for the indicated times were immunoprecipitated with antibodies to CDO (A) or cadherin (pancadherin antibody) (B), fractionated by SDS/PAGE, and immunoblotted with the indicated antibodies. Straight lysates were also probed as indicated. Immunoblotting for myogenin and MHC indicates the progression of cell differentiation. (C) Association of N- and M-cadherin with CDO. (D) Association of BOC, cadherin, and β-catenin in RD cells. (E) Association of cadherin and β-catenin with stably expressed BOC(flag) in C2C12 cells. (F) Association of N- and M-cadherin with stably expressed BOC(flag) in C2C12 cells. For C–F, immunoprecipitation and immunoblotting are as described for A and B.

Colocalization of CDO and BOC with cadherin at sites of cell–cell contact and overlap in C2C12 myoblasts. C2C12 cells were double-stained with anti-flag and anti-pan-cadherin antibodies (Upper) or anti-CDO and anti-pan-cadherin antibodies (Lower). The arrows indicate sites of cell–cell contact and overlap that display enriched colocalization of the indicated proteins. (Scale bar, 25 μm.)

Association of N-cadherin with CDO and BOC via ectodomains and intracellular domains. (A and B) Association of full-length N-cadherin and the intracellular region of N-cadherin with CDO and BOC. 293T cells were transiently cotransfected with expression vectors for CDO (A), BOC(myc) (B), N-cadherin(flag) (A and B), or N-cadherin-intracellular domain [N-cad-ICD(flag)] (A and B). Immunoprecipitation and immunoblotting are as described for Fig. 1. (C) Association of N-cadherin with the ectodomains of CDO and BOC. 293T cells were transiently cotransfected with expression vectors for N-cadherin and CDO-Fc, BOC-Fc, or CD164-Fc. Cell lysates were precipitated with protein A-Sepharose, fractionated by SDS/PAGE, and immunoblotted with the indicated antibodies. Straight lysates were also probed with anti-cadherin antibodies. (D) Cadherin-based adhesion is not required for association of cadherins with CDO. C2C12 cells were cultured on plastic dishes or as a single-cell suspension in the presence of EDTA. Immunoprecipitation and immunoblotting are as described for Fig. 1.

A CDO deletion mutant deficient in associating with N-cadherin interferes with differentiation of C2C12 cells. (A) Schematic diagram of CDO and CDO(ΔFN1). TM, transmembrane. (B) CDO(ΔFN1) is deficient in association with N-cadherin. 293T cells were transiently cotransfected with expression vectors for N-cadherin and CDO or CDO(ΔFN1). Immunoprecipitation (IP) and immunoblotting (WB) are as described for Fig. 1. (C) CDO(ΔFN1) associates normally with CDO-Fc and BOC-Fc. 293T cells were transiently cotransfected with expression vectors for CDO(ΔFN1) and CDO-Fc or BOC-Fc. Cell lysates were precipitated with protein A-Sepharose, fractionated by SDS/PAGE, and immunoblotted with the indicated antibodies. Straight lysates were also probed with anti-CDO antibodies. (D) Stable expression of full-length CDO and CDO(ΔFN1) in C2C12 cells. Western blot (WB) analysis of C2C12 cells infected with pBabePuro-based retroviruses. pBP, cells infected with control retrovirus lacking a cDNA insert; CDO(ΔFN1) and CDO, cells infected with retroviruses harboring the respective mutant and full-length Cdo cDNAs. The blot was probed with antibodies to the CDO intracellular region. (E) Western blot analysis of muscle-specific proteins expressed by the different C2C12 cell infectants cultured under differentiation-inducing conditions for the indicated times. Blots were probed with antibodies to the indicated proteins. (F) Photomicrographs of C2C12 cell infectants cultured in differentiation medium for 3 days, fixed, and stained with an antibody to MHC. The percentage of nuclei in MHC-positive myotubes is indicated. Values are means of triplicate determinations ± 1 SD performed three times. (G) Muscle-specific reporter gene activity. C2C12 cells were transfected in growth medium with the 4Rtk-Luc reporter construct (which is driven by a tetramerized myogenic E-box) and either a control (con) or CDO(ΔFN1)-containing expression vector and then assessed for normalized luciferase activity after 24 h in differentiation medium. Values are means of independent triplicate determinations performed four times.