FIG 4.

Effects of pH on mTORC1 and eEF2K kinase activity. (A) HEK293 cells were incubated at different pHs for 30 min, and mTOR complexes were immunoprecipitated and subjected to mTORC1 activity assay with [γ-³²P]ATP. (B) The mTORC1 activity assay was performed as described for panel A but with nonradiolabeled ATP. (C) eEF2K was immunoprecipitated from lysates of HEK293 cells cultured at pH 7.4 for 30 min, followed by assays for the indicated times. (D) HEK293 cells were cultured at pH 6.6, 7.0, or 7.4 for 30 min, and eEF2K was immunoprecipitated from cell lysates and subjected to activity assays for 10 min. (E) As described for panel D, except that the activity assays were performed for 30 min using nonradiolabeled ATP. For panels A, B, and D, upper panels represent the samples of the lysates (inputs) that were subsequently used in kinases assays. Data shown are representative of three independent experiments.