Figure 9. Bnip-2 and JLP bind the same Cdo complexes, and Bnip-2 regulates p38α/β activity to promote myogenesis. (A) Lysates of COS7 cells transiently transfected with JLP-S, Bnip-2, Cdo, or control (−) expression vectors as indicated were immunoprecipitated (IP) with anti–S agarose and then Western blotted with JLP, Bnip-2, or Cdo antibodies. (B) Lysates of myoblasts of the indicated Cdo genotype cultured in DM for 48 h were immunoprecipitated with Cdo antibodies and then Western blotted with Cdo, Bnip-2, or JLP antibodies. Total cell lysates were also Western blotted with Bnip-2 or JLP antibodies. (C) Lysates of myoblasts of the indicated Cdo genotype cultured in DM for 48 h were immunoprecipitated with Bnip-2 antibodies and then Western blotted with Cdo, Bnip-2, or JLP antibodies. Total cell lysates were also Western blotted with Cdo or JLP antibodies. The Cdo band below the one indicated by the arrow is a partial degradation product. (D) Lysates of C2C12/Bnip-2 (+) or vector control (−) cells in GM or cultured for 48 h in DM were Western blotted with anti–phospho p38α/β (pp38) or p38α/β (p38) antibodies. (E) Lysates of C2C12 cells stably expressing a Bnip-2 siRNA sequence (+) or an irrelevant sequence (−) in GM or cultured for 48 h in DM were Western blotted with pp38 or p38 antibodies. (F) Lysates of C2C12 cells stably expressing a Cdc42Gap siRNA sequence (+) or an irrelevant sequence (−) in GM or cultured for 48 h in DM were Western blotted with pp38 or p38 antibodies. As the lanes were somewhat unevenly loaded in F, the pp38 and p38 signals were quantified by densitometry, and the pp38/p38 ratio reported under each lane in arbitrary units with the control transfectants in GM set to 1. (G) C2C12 cells were cotransfected with control, Bnip-2 siRNA, or Bnip-2 deletion mutant expression vectors, control or MKK6(EE) expression vectors, and pQ-lacZ (a vector that drives expression of nuclear-localized β-gal to mark transfectants). Differentiated cultures were double-stained for β-gal activity (blue) and for MHC expression (brown). Bar, 0.1 mm. (H) Quantification of C2C12 cell differentiation shown in G. Cultures were scored as MHC− or MHC+, with MHC+ cells further scored as having a single (1) nucleus, two to five nuclei, or greater than five nuclei. Values represent means of triplicate determinations ±1 SD. The experiment was repeated three times with similar results.
Figure 9.

Bnip-2 and JLP bind the same Cdo complexes, and Bnip-2 regulates p38α/β activity to promote myogenesis. (A) Lysates of COS7 cells transiently transfected with JLP-S, Bnip-2, Cdo, or control (−) expression vectors as indicated were immunoprecipitated (IP) with anti–S agarose and then Western blotted with JLP, Bnip-2, or Cdo antibodies. (B) Lysates of myoblasts of the indicated Cdo genotype cultured in DM for 48 h were immunoprecipitated with Cdo antibodies and then Western blotted with Cdo, Bnip-2, or JLP antibodies. Total cell lysates were also Western blotted with Bnip-2 or JLP antibodies. (C) Lysates of myoblasts of the indicated Cdo genotype cultured in DM for 48 h were immunoprecipitated with Bnip-2 antibodies and then Western blotted with Cdo, Bnip-2, or JLP antibodies. Total cell lysates were also Western blotted with Cdo or JLP antibodies. The Cdo band below the one indicated by the arrow is a partial degradation product. (D) Lysates of C2C12/Bnip-2 (+) or vector control (−) cells in GM or cultured for 48 h in DM were Western blotted with anti–phospho p38α/β (pp38) or p38α/β (p38) antibodies. (E) Lysates of C2C12 cells stably expressing a Bnip-2 siRNA sequence (+) or an irrelevant sequence (−) in GM or cultured for 48 h in DM were Western blotted with pp38 or p38 antibodies. (F) Lysates of C2C12 cells stably expressing a Cdc42Gap siRNA sequence (+) or an irrelevant sequence (−) in GM or cultured for 48 h in DM were Western blotted with pp38 or p38 antibodies. As the lanes were somewhat unevenly loaded in F, the pp38 and p38 signals were quantified by densitometry, and the pp38/p38 ratio reported under each lane in arbitrary units with the control transfectants in GM set to 1. (G) C2C12 cells were cotransfected with control, Bnip-2 siRNA, or Bnip-2 deletion mutant expression vectors, control or MKK6(EE) expression vectors, and pQ-lacZ (a vector that drives expression of nuclear-localized β-gal to mark transfectants). Differentiated cultures were double-stained for β-gal activity (blue) and for MHC expression (brown). Bar, 0.1 mm. (H) Quantification of C2C12 cell differentiation shown in G. Cultures were scored as MHC− or MHC+, with MHC+ cells further scored as having a single (1) nucleus, two to five nuclei, or greater than five nuclei. Values represent means of triplicate determinations ±1 SD. The experiment was repeated three times with similar results.

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