FIGURE 3.

Neuronal maturation-associated increase of protein synthesis. A, SUnSET Western blot protein synthesis assay. Cultured hippocampal neurons were treated with 20 μg/ml puromycin for 30 min, and nascent peptides were detected using anti-puromycin antibody. Five μg of total protein was used per lane. After normalization to total protein content, levels of puromycinylated peptides reflect relative activity of global protein synthesis during the 30-min labeling period. To provide additional control of total protein content, blots were re-probed with an antibody against GAPDH. Numbers below the blot indicate relative content of nascent peptide signal as determined by densitometry with normalization against the GAPDH signal. Consistent with data in Fig. 1D, expression of GAPDH did not appear to be affected by neuronal maturation when normalized to total protein; however, as expected for rapidly growing cells, total protein content was higher in DIV7 than DIV1 neurons when cell number normalization was applied (data not shown). B and C, neurons were treated with OPP whose incorporation into nascent peptides was visualized using fluorescent Click-iT chemistry. Cycloheximide was added 45 min before OPP. B, representative images of neurons that were identified by co-immunostaining for the neuronal marker NeuN (arrows). B′, translation inhibitor cycloheximide prevented OPP labeling of nascent peptides. Nuclei were visualized by counterstaining with the NuclearMask dye (Invitrogen). C, perikaryal accumulation of nascent peptides kept increasing with neuronal maturation (one-way ANOVA, effect of maturation, F_2,335 = 204.32, p < 0.001). Data represent means ± S.E. of at least 111 cells from two independent experiments, ***, p < 0.001 (post hoc tests).