UCHL1 cosediments with and enhances the assembly of eIF4F. (A) HeLa cells were transduced as shown and, where indicated, pulsed with puromycin, with or without preincubation with cycloheximide. After a 50-minute chase period, lysates were immunoblotted as shown. Each panel represents an independent experiment. (B) Cells expressing the BioID constructs shown were subjected to immunoprecipitation with anti-Myc tag as shown. Lysates, and the resulting precipitates, were probed as shown. The results are representative of at least 2 independent experiments. (C) HeLa cells transduced with either control empty vector lentivirus or wild-type UCH-L1 were subjected to sucrose gradient centrifugation to analyze polysome components, as shown (top), and eluted protein fractions (bottom) were immunoblotted for the indicated proteins. UCH-L1 immunoreactivity corresponds to that of subpolysome complexes containing the eIF4F subunits eIF4E and eIF4A. The results are representative of 2 independent experiments. (D) Pulldowns were performed with m⁷GTP beads and probed as indicated. The loading of precipitates was equalized for the level of eIF4E as it directly binds m⁷GTP. The intensity of retrieved bands was quantitated by normalizing with eIF4E for each condition, using imageJ. Similar results were seen in 3 other cell lines (supplemental Figure 3).