Figure 9

LINC analysis reveals impaired binding of the βH hub domain to CaMKIIβ but not α. (a) Schematic illustration of the LINC assay. Yellow clusters indicate formation of holoenzymes containing WT GFP-CaMKIIα and CIBN-mCh-CaMKII bait. Red clusters indicate inability of monomeric 1–316 truncation to form holoenzymes with CIBN-mCh-CaMKII bait. (b) Representative images of HEK cells expressing full-length GFP-CaMKIIα or the 1–316 truncation that completely lacks the hub domain along with CIBN-mCh-CaMKIIα and CRY2olig. Quantification of LINC imaging reveals co-clustering with CIBN-mCh-CaMKIIα as bait was seen for CaMKIIα and the CaMKIIβe and βeH splice-variant mutants but not for the monomeric 1–316 negative control. Data represent mean ± SEM. n = 5–9 cells. **p < 0.01, ***p < 0.001. Scale bars, 10 µm. (c) Representative images of HEK cells expressing GFP-CaMKIIβe or βeH as well as CIBN-mCh-CaMKIIα and CRY2olig. Quantification of LINC imaging reveals co-clustering with CIBN-mCh-CaMKIIβe as bait was seen for CaMKIIβe but co-clustering was significantly reduced with both the hub-domain lacking CaMKIIβeH variant and the 1–316 negative control. Data represent mean ± SEM. n = 14–28 cells.***p < 0.001. Scale bars, 10 µm.