ExperimentName: PRPF4 physically interacts with PRPF3.

InteractingGene: PRPF3 (HGNC:17348)

InteractionType: Physical

InteractionMethod: Co-immunoprecipitation

InteractionEvidence: Immunoprecipitation of endogenous PRPF3 from HeLa cell nuclear extracts pulled down PRPF4 (Figures 5 and 6).

ExperimentName: BBS7 interacts with other components of the BBSome.

InteractingGene: BBS4 (HGNC:969)

InteractionType: Physical

InteractionMethod: co-immunoprecipitation

InteractionEvidence: Antibody-based isolation of BBS4 results in co-purification of BBS7, as well as BBS1, BBS2, BBS5, BBS8, and BBS9 in stoichiometric quantities. The co-purifying proteins are shown separated by gel electrophoresis in Figure 1F and were identified and quantified by mass spectrometry in Table S1. All 8 proteins are encoded by genes linked to Bardet-Biedl syndrome.

ExperimentName: PDE6A forms a complex with PDE6B and PDE6G

InteractingGene: HGNC:8786 (PDE6B, implicated in retinitis pigmentosa 40) and HGNC:8789 (PDE6G, implicated in retinitis pigmentosa 57)

InteractionType: physical

InteractionMethod: size exclusion chromatography

InteractionEvidence: Purification of the cGMP phosphodiesterase complex from bovine rod outer segments yields a subunit of 88 kDa (PDE6A), a subunit of 84 kDa (PDE6B), and a subunit of 13 kDa (PDE6G), (Figure 2). This article shows that PDE6A interacts with and co-purifies with PDE6B and PDE6G in a single complex isolated from bovine retina. A subsequent article by Pittler et al. later established the position of PDE6A on chromosome 5 (PMID: 2155175).

ExperimentName: Prph2 interacts with Rom1

InteractingGene: ROM1 (HGNC:10254)

InteractionType: Physical

InteractionMethod: Co-immunoprecipitation

InteractionEvidence: Immunoprecipitation of endogenous Prph2 from mouse retinal extracts pulled down Rom1, and vice versa (Fig. 6C).

ExperimentName: RP1L1 physical interaction with RP1

InteractingGene: HGNC:10263 (RP1)

InteractionType: Physical interaction

InteractionMethod: Co-immunoprecipitation

InteractionEvidence: Immunoprecipitation of Rp1 or Rp1l1 from transfected 293T cells successfully led to co-immunoprecipitation of the other, indicating their presence in a complex (Figure 7).

ExperimentName: GPR143 interacts with TYR by co-IP and FRET

InteractingGene: TYR, which is implicated in oculocutaneous albinism types 1A and 1B

InteractionType: Physical

InteractionMethod: Co-immunoprecipitation and FRET were both performed with positive results.

InteractionEvidence: Co-IP of the exogenously expressed GPR143 fusion protein or endogenous TYR found that IP of one protein pulls down the other as well (Figure 2). This was confirmed by fluorescent resonance energy transfer experiments (Figures 4 and 5). Use of a mislocalizing GPR143 mutant control showed that TYR localization appears to be GPR143-dependent (Figures 3 and 4).

ExperimentName: LRP5 interacts with SOST

InteractingGene: SOST (HGNC:13771)

InteractionType: Physical

InteractionMethod: Co-immunoprecipitation

InteractionEvidence: Immunoprecipitation of an exogenously expressed IgG-SOST fusion protein from conditioned medium pulled down the Myc-tagged soluble portion of LRP5 (and LRP6, but not control targets) (Fig. 3A). Subsequent experiments identified SOST as an LRP5/6 antagonist that attenuates canonical Wnt signaling through these receptors (Figure 4).

ExperimentName: NR2E3 physical interaction with NRL and CRX

InteractingGene: HGNC:8002 (NRL), HGNC:2383 (CRX)

InteractionType: Physical interaction

InteractionMethod: Co-immunoprecipitation

InteractionEvidence: Immunoprecipitation of NR2E3, NRL or CRX from bovine retinal nuclear extract successfully led to co-immunoprecipitation of the others, indicating their presence in a transcriptional regulatory complex (Figure 6).

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.