Figure 6

Validation of SILAC-based quantitation by immunoprecipitation and immunoblotting. After treatment of the 6-10B cells with DNP (6 μM), cellular proteins were extracted as described in the methods. Rho-GTPase (A), ferritin (B), LYRIC (C), RNA polymerase (D), and torsin-1A protein 1 (E) were immunoprecipitated from the protein samples by using their corresponding antibodies, and then serine phosphorylation in the immunoprecipitated mixture was detected by immunoblotting using phospho-antibody against serine. The relative immunoblotting ratio of the phosphorylation level of every protein was normalized to its corresponding immunoprecipitated protein. IP, immunoprecipitation. IB, immunoblotting. SILAC, stable isotope labeling by amino acids in cell culture.