Figure 4
Subcellular localization of eIF4B in neurons. (A,B,E,F) Immunofluorescence with antibodies against total (eIF4B-Ct, green; A,E) or Ser504 phosphorylated eIF4B (eIF4B-pS504, green; B,F) and the synaptic proteins Bassoon (red; A,B) or PSD-95 (red; E,F). Total eIF4B shows a diffuse distribution consistent with cytosolic localization, while eIF4B with Ser504 phosphorylation displays a punctate staining localized in neurites. The Color map pictures (right column) show the images of the normalized mean deviation product (nMDP) between two channels, wherein hot colors represent co-localization, and cold colors indicate mutual exclusion. (C,D,G,H) Quantification of the co-localization of eIF4B with Bassoon is expressed by the ICorr score (C,G) and by the average nMDP score (D,H). Data are expressed as mean values ± SEM; n = 30 pictures from three independent experiments. Statistical significance is determined with a Mann-Whitney U test; ***P < 0.0001. (I) Representative western blot of phospho-Ser504 eIF4B (pS504-eIF4B) or total eIF4B in crude brain homogenate (Hom) and synaptosomes (Syn) from adult rat cortex (n = 3). The Syn fraction is enriched in both PSD95 and Ser504 phosphorylated eIF4B. Bar in (A,B,E,F): 10 μm.
