FIG 10.

Monitoring of global mitotic translation by PMY labeling in eIF4G knockout/knock-in cell lines. (A) Time course of DOX-inducible depletion of endogenous eIF4G. (B) HEK293 cell lines with DOX-inducible endogenous eIF4G depletion and myc-eIF4G/eIF4G(Ser1232Ala) reconstitution were induced for 3 days. During the last day, the cells were G₂ arrested with Ro3306 and released to NOC-containing growth medium for mitotic arrest. (C) Time course of PMY incorporation into nascent polypeptide chains. Exponentially growing HEK293 cells were pretreated with DMSO or 10 μM AMY for 15 min and incubated with 5 μM PMY. To stop puromycylation, cells were washed with cold PBS at 4°C and lysed. Lysates were subjected to immunoblotting with the indicated antibodies. The quantitative data represent an average value of PMY/GAPDH ratio from three independent experiments. Standard errors in all cases were less than 12% from the average value. (D) The effect of Ser1232Ala substitution in eIF4G on global mitotic translation (samples from assays shown in panel B were tested). Nonsynchronized (NS) cells and cells arrested in G₂ and M phases were treated with 5 μM PMY for 15 min. The reaction was stopped, cells were lysed, and the lysates were subjected to PMY immunoblotting. The assay was repeated in three independent series, each consisting of three separate samples. The PMY/GAPDH ratio was quantified.