p104 Binds to Rac1 and Reduces Its Activity during Myotube Differentiation of C2C12 Cell
Figure 4
The interaction of p104 with CrkII decreased the activity of Rac1. (a) NIH3T3 cells were transfected with empty vector (Mock) or p104 constructs. After 24 h of transfection, the amount of GTP-bound (active) Rac1 was determined by GST-PAK pull-down analysis, followed by Western blotting with an anti-Rac1 antibody (upper panel). 50 μg of cell lysate was used for Western blot analysis with an anti-Rac1 antibody to check the total amount of Rac1 (middle panel). The amount of GST-PAK used in this assay was assessed by coomassie brilliant blue staining (bottom panel). (b) NIH3T3 cells were transfected with GFP-tagged full-length p104 (FL), second proline-rich motif mutant (2mp) and carboxy-terminal deletion mutant (C). The level of GTP-bound Rac1 was measured by GST-PAK pull-down analysis, followed by Western blotting with anti-Rac1 antibody. (c) Lysates from NIH3T3 cells transfected with GFP-tagged full-length p104, 2mp mutant, and carboxy-terminal deletion mutant were immunoprecipitated with an anti-GFP antibody, and then Western blot analysis was performed with anti-CrkII (upper panel) and anti-Rac1 antibodies (lower panel). (d) Inhibition of JNK activity by p104 overexpression. To measure the JNK activity, cells were serum-starved for 12 h and then treated with 5 μM PMA/ionomycin for 5 min. JNK was immunoprecipitated and kinase assays were performed using GST c-Jun as a substrate (upper panel). After treatment with 50 ng/mL PDGF BB, 50 μg of each cell lysate was separated on SDS-PAGE gel and they were analyzed by Western blot using an anti-active ERK antibody to check the Erk activity (lower panel).
