Figure 2.
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).

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).

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