ExperimentName: Co-IP showing interaction of RPGR with CEP290

InteractingGene: CEP290, which is implicated in Leber congenital amaurosis 10 and multiple syndromic phenotypes (Joubert syndrome, Meckel syndrome, and Senior-Loken syndrome).

InteractionType: Physical

InteractionMethod: Co-immunoprecipitation was performed with a positive result, while yeast-2-hybrid (Y2H) was performed with a negative result.

InteractionEvidence: Co-IP with an RPGR antibody (Figure 4A) or CEP290 antibody (Figure 4B) successfully immunoprecipitates both proteins from wild-type mouse retinal extracts. The two (endogenously expressed) proteins also co-localize in IMDC-3 cells (Figure 4C). However, yeast 2-hybrid experiments do not confirm this interaction as direct (data not shown).

ExperimentName: GUCY2D protein-protein interaction with RD3

InteractingGene: HGNC:19689 (implicated in LCA12)

InteractionType: Physical (protein-protein) interaction

InteractionMethod: Interaction was established by coimmunoprecipitation followed by mass spectrometry (Table S1), confirmed by western blotting (Figure 2A).

InteractionEvidence: Mouse Rd3 was immunoprecipitated by monoclonal antibody and co-purifying proteins were identified by LC-MS/MS. The highest-scoring interacting partner was Gucy2e, the mouse ortholog of GUCY2D (Table S1). This was confirmed by western blotting (Figure 2A) and found to be dependent on the C-terminus of Gucy2e (Figure 5).

ExperimentName: Interaction with TPP1 and CLN3

InteractingGene: TPP1 (formerly known as CLN2) and CLN3

InteractionType: Physical (protein-protein) interaction

InteractionMethod: Co-immunoprecipitation

InteractionEvidence: PPT1 (aka CLN1), CLN3, and CLN5 were exogenously expressed in COS-1 cells. Immunoprecipitation of endogenous TPP1 (Fig. 5A) or exogenously expressed CLN3 (Fig. 5B) successfully co-immunoprecipitated CLN5 (demonstrated by western blotting). Immunoprecipitation of GST-CLN5 successfully co-immunoprecipitated TPP1 and CLN3 (Fig. 5C). This evidence helps validate the link between CLN5 variants and neuronal ceroid lipofuscinosis, as both TPP1 and CLN3 have also been linked with this disease state.