FIGURE 7.

Effect of amino acid withdrawal on [³²P]guanyl nucleotide content of RagC-containing heterodimers endogenous to ³²P_i-labeled HEK293T cells. Replicate plates of HEK293T cells were incubated in P_i-free DMEM containing ³²P_i (0.2 mCi/ml). After 4 h, the cells were rinsed and incubated in either homemade P_i-free medium (AA+) or P_i-free medium lacking amino acids (AA-), each containing ³²P_i (0.2 mCi/ml). Cells were extracted 2 h later, and immunoprecipitation (IP) was performed using nonimmune (NI) rabbit IgG or anti-RagC IgG. Nucleotides were extracted from the washed immunoprecipitates and separated by TLC on PEI cellulose. An immunoblot analysis of the extract for 4E-BP(T37P/T46P) and of the immunoprecipitates for RagC are shown in the right panels. Ori, origin.