Figure 1.

Interaction and co-localization of CPEB-containing cytoplasmic complex proteins. (A,B) Mouse whole brain lysates were immunoprecipitated with antibodies against symplekin, Ngd, or IgG control and immunoblotted as indicated. (C, D) Lysates from HEK293 cells expressing Gld2 or vector only or neuroblastoma cells expressing CPEB-GFP, Gld2-GFP, or GFP were used for co-immunoprecipitation with symplekin antibody or IgG control. (E, F) FLAG antibodies were used for immunoprecipitation of cell lysates from neuroblastoma cells expressing: (E) CPEB-FLAG or FLAG and PARN-GFP, CPEB-GFP, or GFP, and (F) PARN-FLAG or FLAG and CPEB-GFP or GFP, followed by immunoblotting for FLAG, GFP, symplekin, and tubulin. (G) Hippocampal neurons (17 DIV) were co-immunostained for symplekin (red) and CPEB, Gld2, PARN, Ngd, or GluR1 (green). The images were deconvolved and analyzed for dendritic 3D co-localization. Pixels with overlapping signals are shown in white; the scale bar is 10 μm. (H) The probability of non-random co-localization was determined for symplekin and CPEB, Gld2, PARN, Ngd or GluR1. The error bars refer to SEM. (I) Mander’s overlap coefficients were computed for each co-localized pair. The error bars refer to SEM. (J) High-magnification images and 3D reconstructions depict localization of symplekin (red) and CPEB, Gld2, PARN or Ngd (blue) in dendritic spines (green: phalloidin; scale bar is 0.5 μm). (see Figure S1)