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1.

Figure 4. Localization of SMC1 and SMC3 to primary cilia. From: RPGR-ORF15, which is mutated in retinitis pigmentosa, associates with SMC1, SMC3, and microtubule transport proteins.

A: Immunogold labeling of mouse retina using SMC1 (1:300) and SMC3 (1:50) antibodies. The labeling (arrowheads) of the connecting cilium (CC) by the SMC3 antibody is quite robust. The labeling by the SMC1 antibody is much less, but still significant. Scale bars: 300 nm.
B: MDCK cells were stained with acet α-tubulin (red), anti-SMC1 or anti-SMC3 (green) antibodies. Merge (yellow) shows punctate staining pattern (depicted by arrowheads) for SMC1 and SMC3 along the axoneme of the primary cilia. Negative control did not show a staining (data not shown).

Hemant Khanna, et al. J Biol Chem. ;280(39):33580-33587.
2.

Figure 7. Expression of RPGR-ORF15 in the retina of Rpgr-knockout mouse. From: RPGR-ORF15, which is mutated in retinitis pigmentosa, associates with SMC1, SMC3, and microtubule transport proteins.

A: Retinal protein extract (50 μg each) from wild-type (wt) and Rpgr-knockout (ko) mouse (24) was analyzed by SDS-PAGE, followed by immunoblotting using the ORF15CP, CT-15, GR-P1, and β-tubulin (as control for protein loading) antibodies. Lanes are as indicated. Arrows indicate RPGR-ORF15 isoform(s) and asterisk (*) the constitutive isoform.
B: Immunogold labeling of the Rpgr-knockout mouse retina using the ORF15CP antibody. The antibody labels the connecting cilium (CC) as well as basal bodies (BB) of photoreceptors. Scale bar: 300 nm.

Hemant Khanna, et al. J Biol Chem. ;280(39):33580-33587.
3.
Figure 2

Figure 2. Association of RPGR-ORF15 isoform(s) with SMC1 and SMC3. From: RPGR-ORF15, which is mutated in retinitis pigmentosa, associates with SMC1, SMC3, and microtubule transport proteins.

A: Localization of RPGR-ORF15, SMC1, and SMC3 in different subcellular fractions of bovine retina. Total bovine retinal extract (BR), cytosol, detergent soluble (Det. Sol.) and axoneme (Ax) fractions of the retina were analyzed by SDS-PAGE and immunoblotting using indicated antibodies. Molecular weight markers in kDa are shown on the left.
B: Co-immunoprecipitation of SMC1 and SMC3 with RPGR-ORF15 from bovine retinal axoneme. Axoneme-enriched fraction (250 μg) was immunoprecipitated using the ORF15CP antibody. The proteins were analyzed by SDS-PAGE followed by immunoblot (IB) analysis using SMC1 or SMC3 antibodies. Lanes are as indicated. * indicates IgG heavy chain. Molecular weight markers in kDa are shown on the left of each panel.

Hemant Khanna, et al. J Biol Chem. ;280(39):33580-33587.
4.
Figure 6

Figure 6. Dynein-dependent localization of RPGR-ORF15 to basal bodies. From: RPGR-ORF15, which is mutated in retinitis pigmentosa, associates with SMC1, SMC3, and microtubule transport proteins.

ARPE-19 cells were transiently-transfected with the p50-dynamitin expression construct, followed by immunocytochemistry, as described in Experimental Procedures. Only transfected cells expressing p50-dynamitin show red cytoplasmic fluorescence. The localization of RPGR (small thin arrows), ninein (large thicker arrows), and/or γ-tubulin (arrowheads) is indicated in cells that do (with red fluorescence) or do not express the p50-dynamitin construct. The basal body localization of RPGR (green) is detectable in untransfected cells that do not overexpress p50-dynamitin. The p50-overexpressing cells (red) do not exhibit basal body localization of RPGR or ninein, as evident in the merged view. Localization of γ-tubulin to basal bodies was unaffected in these cells. Similar results were obtained in IMCD3 cells (data not shown).

Hemant Khanna, et al. J Biol Chem. ;280(39):33580-33587.
5.
Figure 5

Figure 5. Co-immunoprecipitation of basal body and microtubule-associated proteins with RPGR-ORF15. From: RPGR-ORF15, which is mutated in retinitis pigmentosa, associates with SMC1, SMC3, and microtubule transport proteins.

A: Immunoprecipitation (IP) using the ORF15CP antibody or normal rabbit IgG from the bovine retinal axoneme fraction (250 μg) was performed. The proteins were analyzed by SDS-PAGE followed by immunoblotting using the indicated antibodies below each panel. Lanes are: 1, Input (retinal axoneme extract); 2, IP using ORF15CP; 3, IP using normal IgG.
B: Reverse IP using the indicated antibodies was performed and analyzed by SDS-PAGE and immunoblotting. The immunoblot was probed with ORF15CP antibody. Arrow indicates faint ORF15CP immunoreactive bands immunoprecipitated with the anti-p50dynamitin antibody. Ax: Axoneme fraction. Molecular weight markers in kDa are shown on the left.

Hemant Khanna, et al. J Biol Chem. ;280(39):33580-33587.
6.

Figure 1. Localization of RPGR-ORF15 isoform(s) to primary cilia. From: RPGR-ORF15, which is mutated in retinitis pigmentosa, associates with SMC1, SMC3, and microtubule transport proteins.

A: Immunogold labeling of human and mouse retina using the ORF15CP antibody. The signal is concentrated in the connecting cilium (CC) and basal bodies (BB). Scale bars: 300 nm. B: Left panel: Staining of mouse sperm flagellum with ORF15CP (green) and anti-acet α-tubulin (red). Merged image shows co-localization of the two signals (yellow) along the length of the axoneme and tip of the flagellum. Blue color in the head of sperm shows DAPI staining for nuclei. Right panel: RPGR-ORF15 (green) co-localizes with acet α-tubulin (red) in MDCK cells in a punctate pattern (Merge).

Hemant Khanna, et al. J Biol Chem. ;280(39):33580-33587.
7.
Figure 3

Figure 3. Interaction of RPGR-RLD with SMC1 and SMC3. From: RPGR-ORF15, which is mutated in retinitis pigmentosa, associates with SMC1, SMC3, and microtubule transport proteins.

A: GST pulldown assay: GST-RLD fusion protein or GST alone (5 μg) was incubated with bovine retinal axoneme (250 μg) followed by addition of glutathione-Sepharose beads. SMC1 and SMC3 bound to GST-RLD but not GST moiety. Lanes are as indicated. Molecular weight markers in kDa are shown on the left. IB: Immunoblot.
B: Immunoprecipitation from transiently transfected MDCK cells: MDCK cells were transfected with construct encoding either Xpresss (Xp) tag alone or a Xp-tagged mRPGR-C1 protein. After transfection, cells were lysed and protein extract was subjected to immunoprecipitation followed by SDS-PAGE and immunoblot (IB) analysis using indicated antibodies. Both SMC1 and SMC3 displayed specific binding to mRPGR-C1.
C: In vitro transcription/translation and co-IP: Interaction between Xp-mRPGR-C1 and SMC1, SMC3 and phosphorylation mutants of SMC1 was studied by synthesizing the 35S-labeled or unlabeled proteins in vitro followed by immunoprecipitation (IP) as described in Experimental procedures.
Left panel: Non-radiolabeled Xp-mRPGR-C1 and 35S-labeled SMC1, SMC3, and various mutants of SMC1 were used and IP was performed using anti-Xp antibody. The immunoprecipitated proteins were analyzed by SDS-PAGE and autoradiography. Lanes are as indicated. Molecular weight markers in kDa are shown on the left.
Right panel: 35S-mRPGR-C1 was detected when non-radiolabeled SMC1, SMC1 S957A, and SMC3 were used for immunoprecipitation. All constructs synthesized equal amount of proteins as determined by visual estimation of the inputs analyzed by SDS-PAGE followed by autoradiography (data not shown). Immunoprecipitation using normal IgG did not show a signal (data not shown).

Hemant Khanna, et al. J Biol Chem. ;280(39):33580-33587.

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