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. 2003 Mar 12;100(7):3989–3994. doi: 10.1073/pnas.0736565100

Figure 4.

Figure 4

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.