p104 Binds to Rac1 and Reduces Its Activity during Myotube Differentiation of C2C12 Cell

Figure 5

Interaction of p104 with Rac1 and CrkII during C2C12 differentiation. (A) C2C12 cells were grown to confluence (a) and then placed in differentiation medium for 5 days to induce the differentiation into myotube formation (b). (B) The activity of Rac1 was decreased during C2C12 differentiation. The amount of GTP-bound Rac1 in cell lysates prepared from undifferentiated (Undif.) or differentiated (Dif.) myoblasts was analyzed by GST-PAK pull-down analysis. Rac1-GTP (active form) and total Rac1 from the same extracts were detected by Western blotting with Rac1 antibody. (C) Transcript levels of p104, myogenin, and α-actin and the protein level of p104 were determined in the absence (Undif.) or presence (Dif.) of differentiation medium by reverse transcription-polymerase chain reaction and Western blot analysis, respectively. The mRNA levels of p104 and differentiation markers were increased 5 days after the induction of differentiation. Levels of glyceraldehydes-3-phosphate dehydrogenase (GAPDH) transcript and actin were used as a control. (D) Interaction of p104 with Rac1 and CrkII was increased during the C2C12 differentiation. C2C12 cells were grown in differentiation media for 5 days and total protein was extracted. Lysates from undifferentiated or differentiated cells were immunoprecipitated with p104 antiserum, and then Western blot analysis was performed using anti-CrkII, anti-Rac1, and anti-p104 antibodies. (E) The effect of p104 overexpression in the myotube differentiation. The C2C12 cells were transfected with p104 ((i)–(p)) and grown in the presence ((e)–(h), (m)–(p)) or absence ((a)–(d), (i)–(l)) of differentiation media. The photos were taken at the indicated days after the addition of differentiation media.