Figure 4. eIF2β phosphorylation stimulates TC recycling.

(a) HEK293E cells that stably express exogenous WT or indicated FLAG-eIF2β mutants in which endogenous eIF2β was depleted by shRNA (Supplementary Fig. 4b; Methods) were serum starved for 16 h, stimulated with serum (10%) for 30 min and subjected to FLAG immunoprecipitation (IP). Inputs (10%; Supplementary Fig. 4c) and quantity of immunoprecipitated proteins (25%) was determined by western blotting. The amount of tRNA_i^Met in the immunoprecipitated material was monitored by semi-quantitative reverse transcriptase–PCR (sqRT–PCR). tRNA^Lys was used as a negative control. (b) Cells described in a that express WT or S(2,67)D FLAG-eIF2β were serum starved for 16 h and then stimulated with 10% serum (fetal bovine serum, FBS) in the presence of a vehicle (DMSO), rapamycin (50 nM) or torin1 (250 nM) for 30 min. Lysates were immunoprecipitated with an anti-FLAG antibody. The quantity of tRNA_i^Met and tRNA^Lys in immunoprecipitates and input (10%) was analysed by sqRT–PCR, whereas the levels of indicated protein were determined by western blotting. (c) Cells described in (a) were serum starved for 16 h and then stimulated with 10% serum (FBS) for 30 min in the presence of a vehicle (DMSO) or torin1 (250 nM). Immunoprecipitations were carried out as described in a. The amount of indicated proteins in FLAG-eIF2β immunoprecipitated material and inputs (10%) was monitored by western blotting. (b,c) Western blotting experiments were performed in independent duplicates and the representative results are shown. sqRT–PCR (n=3) results were independently confirmed using quantitative RT–PCR (Supplementary Fig. 4d,e).