FIG. 4.
Overexpression of HBP1 inhibited C2C12 differentiation in a transient-differentiation assay. A transient-differentiation assay was devised to score the effects of HBP1 on C2C12 differentiation. The basis of the assay was that expression of MyoD family members could accelerate C2 cell differentiation. (A) C2C12 cells grown on coverslips were transfected with plasmids encoding wild-type or mutant HBP1, Myf5, and β-Gal. After 24 h, the transfected cells were cultured in medium supplemented with 2% FCS for another 40 h. Cells were fixed and immunostained for β-Gal and MHC. The percentage of differentiated cells (MHC positive) among the transfected cells (β-Gal positive) was determined. Each transfection was repeated three times, and cells on two different coverslips from each experiment were counted. The total number of cells counted for each experiment was 200 to 300. All lanes contain β-Gal and the following additions: lane 1, Myf5 (filled column); lane 2, Myf5 and wild-type HBP1 (vertical stripes); lane 3, Myf5 and HA-ΔHBP403-513 (open column); and lane 4, no addition (horizontal stripes). (B) To measure protein expression levels, the lysates from a parallel experiment were analyzed for the expression of HA-Myf5, HA-ΔHBP403-513, and HA-HBP1 by using immunoprecipitation with anti-Myf5 or anti-HBP1 antibodies, respectively, followed by Western blot analyses of immune complexes with the anti-HA antibody. The constructs and antibodies are described in Materials and Methods. A representative experiment is shown. (Ba) Western blot showing Myf5 expression. The assay was performed as described for panel B. Cells were transfected with β-Gal and Myf5 (lane 4), Myf5 and HBP1 (lane 3), Myf5 and ΔHBP403-513 (lane 2), or no other expression vector (lane 1). (Bb) Western blot showing HBP1 expression. Cells were transfected with β-Gal, Myf5 and HBP1 (lane 1) or Myf5 (lane 2). (BC) Cells were transfected with β-Gal and Myf5 and ΔHBP403-513 (lane 1) or Myf5 (lane 2).