Fig. S2.

MCP is associated with endogenous β-actin mRNA in MBS cells. (A–C) Representative smFISH-IF images in WT neurons (control): dissociated hippocampal neurons derived from WT mice (A and B) expressing or (C) not expressing MCP-GFP were probed for IF for MCP-GFP (GFP antibody; green) and smFISH using the following FISH probes: (A) MBS probes (Cy3; red) and (B and C) β-actin ORF probes (Cy3; red). In WT neurons, β-actin mRNA did not have MBS in its 3′-UTR; thus, MCP-GFP did not bind the mRNA, and it is retained in the nucleus because of a nuclear localization signal signal. (A) No discrete fluorescent signal was detected in either channel. (B and C) Only fluorescent spots in smFISH channel were detected using β-actin ORF probes. MAP2 is shown in blue as a dendrite marker. Images are representative of two independent experiments, with over 20 dendrites observed in each experiment. (Scale bars: 10 μm.) (D) Distribution of observed distances for MCP-MBS (<50 nm; gray bars) and MCP-CaMKII (>150 nm; black bars). The higher observed distances between MCP and CaMKII mRNA suggest a random association. (E and F) Scatterplots showed the probability of chance association between molecules for (E) MCP-GFP and β-actin mRNA (MBS) in MCP-MBS and (F) MCP-GFP and CaMKII mRNA (CaMKII) in MCP-CaMKII. Boxes A and B are expanded in Fig. 2 F and G, respectively, for better visualization. (G and H) Histograms of signal intensity for (G) MCP and (H) MBS. Gray bars indicate total population, and red bars indicate physically associated mRNA and protein molecules defined by box A.