Figure 3.

Pum2 promotes eIF4E translation. (A,B) RNA (A) and protein (B) fold changes of growth regulators upon knock-down of Pum2 (data extracted from the work in [18]). (C) Consensus sequence and E-value of the 5′-UTR of Pum2 regulated mRNAs identified by MEME analysis. (D,E) Representative immunoblot (D) against eIF4E and quantifications I upon incubation of mature (14 DIV) cortical neurons with bFGF, NGF, and EGF, respectively, for two days. β-III Tubulin was used as loading control. (F) Representative immunoblot against eIF4E from shControl and shPum2 transduced cortical neurons (14 DIV) and quantification normalized to Vinculin and β-III Tubulin. (G) Relative eIF4E mRNA levels in shControl and shPum2 transduced neurons. Peptidyl-prolyl cis-trans isomerase A (PPIA) was used as reference gene. Line connects control and Pum2 depleted conditions from identical cultures. (H) Representative polysome profile (18–50%) of post-nuclear brain lysate and representative immunoblots for Pum2 (eIF4E and PABPC1 served as marker for the translation initiation machinery, Rpl7a for ribosomes). (I) Representative immunoblot for Pum2 upon centrifugation through high-resolution sucrose gradients (5–25%). eIF2s1 and eIF4E served as markers for the translation initiation machinery, Rpl7a and Rps6 for large and small ribosomal subunits, respectively, FMRP is a Pum2 protein interactor. (J) Representative immunoblot against eIF4E, PAPBC1, and Pum2 upon incubation with m⁷G beads. Arrow heads indicate the respective proteins. p-values were calculated using Wald test (A), unpaired Student’s t-test (B), and one-sample t-test (E,F). * p < 0.05, ** p < 0.01, **** p < 0.0001, 3–4 biological replicates.