Novel CAR Exon 8 Interactions pull PICK1-GFP to the intercellular junctions. Co-localization of hCAREx8 and PICK1-GFP in the perinuclear region may represent an artifact of high protein expression or alternatively PICK1-related retention of CAREx8. hCAREx8 interaction with MAGI-1b-GFP results in hCAREx8 degradation hCAREx8 differentially interacts with hCAREx7 PDZ-mediated interacting partners despite having a similar class of PDZ binding domain. We have previously shown an interaction between hCAREx7 and MAGI-1b-GFP (Figure 4A-C). To investigate the interaction between hCAREx8 and MAGI-1b-GFP, COS-7 cells were co-transfected and evaluated by immunocytochemistry. Surprisingly, little to no hCAREx8 staining (Figure 4D) was present in cells expressing MAGI-1b-GFP (Figure 4E). Most of the small amount of hCAREx8 present within MAGI-1b-GFP positive cells appeared within vesicular structures and not the junctions (Figure 4F). The presence, localization and amount of hCAREx8 appeared to be dependent on the amount of MAGI-1b-GFP. A small percentage of cells appeared to express hCAREx8 alone and the expression was robust in comparison to neighboring MAGI- 1b-GFP expressing cells (Figure 4G-I).This was not the case for Figure 4. Co-expression of hCAREx8 and MAGI-1b-GFP results in the loss of hCAREx8. In contrast to the co-localization of hCAREx7 (A, red) and MAGI-1b-GFP (B, green) as shown in panel C (yellow), co-expression of hCAREx8 (D, G, red) and MAGI-1b-GFP (E, H, green) results in decreased levels of hCAREx8 (F) unless MAGI-1b-GFP is absent from the cell (I). Co-expression of hCAREx8 (J, red) with GFP (K, green) results in abundant hCAREx8 expression at the junctions of the cells and diffuse GFP expression (L). Confocal microscopy (60x oil immersion). doi:10.1371/journal.pone.0009909.g004 PLoS ONE | www.plosone.org 5 March 2010 | Volume 5 | Issue 3 | e9909