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. 2012 May 23;32(21):7119–7136. doi: 10.1523/JNEUROSCI.0784-12.2012

Figure 6.

Figure 6.

Palmitoylation controls AKAP79 assembly of signaling complexes containing PKA, CaN, and SAP97 at endosomes. A, COS7 cells expressing 79WT or C36,129S-CFP (blue) and PKA-RIIα-YFP (green) fixed and immunostained for EEA1 (red) reveals plasma membrane colocalization of AKAP79 and PKA (turquoise in merge panels) and endosome colocalization of all three proteins (insets) for 79WT (white in merge panels) but only plasma membrane AKAP79/PKA colocalization for C36,129S. B, Quantification of percentage of EEA1 fluorescence colocalized with AKAP79-CFP or PKA-RII-YFP for 79WT (79-CFP, 38 ± 5%; PKA-RII-YFP, 54 ± 10%) and C36,129S (79-CFP, 4 ± 2%; PKA-RII-YFP, 4 ± 1%; ***p < 0.001 by t test) reveals loss of PKA endosome targeting for C36,129S. n = 6–7 cells. C, COS7 cells expressing 79WT or C36,129S-CFP (blue) and CaNAα-YFP (green) fixed and immunostained for EEA1 (red) reveals plasma membrane colocalization of AKAP79 and CaN (turquoise in merge panels) and endosome colocalization of all three proteins (insets) for 79WT (white in merge panels) but only plasma membrane AKAP79/CaN colocalization for C36,129S. D, Quantification of percentage of EEA1 fluorescence colocalized with AKAP79-CFP or CaNA-YFP for 79WT (79-CFP, 46 ± 12%; CaNA-YFP, 48 ± 15%) and C36,129S (79-CFP, 7 ± 2%; CaNA-YFP, 6 ± 2%; ***p < 0.001 by t test) reveals loss of CaN endosome targeting for C36,129S; n = 5–9 cells. E, COS7 cells expressing 79WT or C36,129S-CFP (blue) and βSAP97i3-YFP (green) fixed and immunostained for EEA1 (red) reveals plasma membrane colocalization of AKAP79 and SAP97 (turquoise in merge panels) and endosome colocalization of all three proteins (insets) for 79WT (white in merge panels) but mainly plasma membrane AKAP79/SAP97 colocalization for C36,129S. F, Quantification of percentage of EEA1 fluorescence colocalized with AKAP79-CFP or SAP97i3-YFP for 79WT (79-CFP, 43 ± 14%; SAP97i3-YFP, 43 ± 14%) and C36,129S (79-CFP, 6 ± 2%; SAP97i3-YFP, 17 ± 2%; *p < 0.05, **p < 0.01 by t test) reveals reduced SAP97 endosome targeting for C36,129S; n = 7–10 cells. Error bars indicate SEM.