Journal: PLoS ONE

Article Title: SDCCAG8 Interacts with RAB Effector Proteins RABEP2 and ERC1 and Is Required for Hedgehog Signaling

doi: 10.1371/journal.pone.0156081

Figure Lengend Snippet: ( A , B ) Sdccag8 gt/gt cells have shorter cilia. Immunofluorescence staining of cultured mouse embryonic fibroblasts derived from wild type ( A ) and Sdccag8 gt/gt ( B ) mice using antibodies against distal appendage marker CEP164 (red) and cilia marker acetylated tubulin (green), demonstrate shorter cilia in Sdccag8 gt/gt cells ( B ). ( C ) Quantitation of the percentage of ciliated cells for wild type and Sdccag8 gt/gt cells after serum starvation (48 hrs). Significantly less Sdccag8 gt/gt cells grow cilia compared to wild type cells (18% vs. 93%, n = 100 for both genotypes, **p = 0.0018). ( D ) Sdccag8 gt/gt MEFs have significantly shorter cilia (1.9 ± 0.2 μm, n = 20) compared to wild type cells (2.9 ± 0.0 μm, n = 142, ****p<0.0001). ( E ) Sdccag8 gt/gt MEFs have attenuated response to Hh signal agonist SAG. qRT-PCR analysis demonstrates reduced levels of Hh pathway target gene Gli1 in SAG treated Sdccag8 gt/gt MEFs compared to wild type cells (N = 3). ( F ) Knockdown of SDCCAG8 causes a reduction in cilia formation in hTERT-RPE1 cells. Only 43% of SDCCAG8 knockdown cells grew cilia compared to 94% wild type cells (**p = 0.0092). ( G ) Cilia length is significantly reduced in SDCCAG8 knockdown cells (1.9 ± 0.1 μ, n = 63) compared to wild type cells (2.8 ± 0.1 μ., n = 80, ****p<0.0001). Scale bar: ( A , B ) 7.5 μm. wt , Sdccag8 wild type allele; gt , Sdccag8 gene-trap allele. ( H , I , J , K ) Immunofluorescence staining of control siNS hTERT-RPE1 cells ( H , J ) and siSDCCAG8 hTERT-RPE1 cells ( I , K ) with antibodies against acetylated tubulin (green) and distal appendage markers CEP83 (red) ( H , I ) or FBF1 (red) ( J , K ), reveals no abnormalities in the formation of centriolar distal appendages despite the loss of cilia in SDCCAG8 knockdown cells ( I , K ) compared to control cells transfected with non-specific siRNA ( F , H ).

Article Snippet: Primary antibodies used were as follows: rabbit polyclonal anti-CEP164 was a gift from Erich Nigg (University of Basel, Switzerland), rabbit polyclonal anti-ERC1 (A302-698A, Bethyl Laboratories), rabbit polyclonal anti-CEP131 (ab99379, Abcam), rabbit polyclonal anti-RABEP2 (ab138279, Abcam), mouse monoclonal anti-SDCCAG8 (ab67098, Abcam), rabbit anti-DYKDDDDK Tag (#2368, Cell Signaling), mouse monoclonal anti-γ-tubulin (GTU-88, Sigma), rabbit anti-acetylated α-tubulin (#5335, Cell Signaling), mouse monoclonal anti-polyglutamylated tubulin (T9822, Sigma-Aldrich), rabbit polyclonal anti-CCDC41 (CEP83) (HPA038161, Atlas), rabbit polyclonal anti-FBF1 (HPA023677, Atlas), chicken anti-GFP (GFP-1020, Aves).

Techniques: Immunofluorescence, Staining, Cell Culture, Derivative Assay, Marker, Quantitation Assay, Quantitative RT-PCR, Transfection

Journal: PLoS ONE

Article Title: SDCCAG8 Interacts with RAB Effector Proteins RABEP2 and ERC1 and Is Required for Hedgehog Signaling

doi: 10.1371/journal.pone.0156081

Figure Lengend Snippet: ( A ) Co-staining with the centriolar protein γ-tubulin demonstrates centrosomal localization for the identified SDCCAG8 interacting proteins RABEP2, ERC1, and CEP131 in hTERT-RPE1 cells. Note CEP131 localization also at centriolar satellites. Scale bars: 5 μm. ( B ) Co-staining with the ciliary axoneme marker poly-glutamylated tubulin (red) demonstrates ciliary and basal body localization of RABEP2, basal body localization of ERC1 and centriolar satellite localization of CEP131 in hTERT-RPE1 cells. Scale bars: 5 μm. ( C ) Quantification of the co-localization coefficients of RABEP2, ERC1 and CEP131 with γ-tubulin positive centrosomes in cells from ( A ). In each case n>40 centrosomes; error bars, SEM. ( D ) Quantification of the co-localization coefficients of RABEP2, ERC1 and CEP131 with polyglutamine-tubulin positive centrosomes in cells from ( B ). In each case n>40 centrosomes; error bars, SEM.

Article Snippet: Primary antibodies used were as follows: rabbit polyclonal anti-CEP164 was a gift from Erich Nigg (University of Basel, Switzerland), rabbit polyclonal anti-ERC1 (A302-698A, Bethyl Laboratories), rabbit polyclonal anti-CEP131 (ab99379, Abcam), rabbit polyclonal anti-RABEP2 (ab138279, Abcam), mouse monoclonal anti-SDCCAG8 (ab67098, Abcam), rabbit anti-DYKDDDDK Tag (#2368, Cell Signaling), mouse monoclonal anti-γ-tubulin (GTU-88, Sigma), rabbit anti-acetylated α-tubulin (#5335, Cell Signaling), mouse monoclonal anti-polyglutamylated tubulin (T9822, Sigma-Aldrich), rabbit polyclonal anti-CCDC41 (CEP83) (HPA038161, Atlas), rabbit polyclonal anti-FBF1 (HPA023677, Atlas), chicken anti-GFP (GFP-1020, Aves).

Techniques: Staining, Marker

Journal: PLoS ONE

Article Title: SDCCAG8 Interacts with RAB Effector Proteins RABEP2 and ERC1 and Is Required for Hedgehog Signaling

doi: 10.1371/journal.pone.0156081

Figure Lengend Snippet: (A) Western blot analysis of extracts from control or siRABEP2-depleted hTERT-RPE1 cells, probed with anti-RABEP2 or anti-β-actin, as a loading control. Dharmacon SMARTpool siRNAs were used to knock down endogenous RABEP2 at high efficiency. (B) RABEP2 knockdown abolishes cilia formation in hTERT-RPE1 cells. Control, RABEP2 and SDCCAG8 depleted hTERT-RPE1 cells were serum starved for 48 hours after transfection with siRNA, fixed, and immunostained for acetylated α-tubulin to label primary cilia. Percentage of ciliated cells was determined in each case, control 92.8 ± 5.2%, n = 50; siRABEP2 15 ± 8.6%, n = 50 and siSDCCAG8 31.48 ± 1.694%, n = 50. Error bars, SEM, ****p<0.0001. (C) hTERT-RPE1 cells were transfected with non-specific siRNA (siNS), siRABEP2, or siSDCCAG8. Cells were ciliated by serum starvation and stained with antibodies against SDCCAG8 (green) and acetylated α-tubulin (red). Knockdown of RABEP2 abolishes cilia formation, but does not alter SDCCAG8 localization at the centrioles. Knockdown of SDCCAG8 abolishes cilia formation in hTERT-RPE1 cells. Scale bars: 5 μ m. (D) hTERT-RPE1 cells were transfected with non-specific siRNA (siNS), siRABEP2, or siSDCCAG8. Cells were ciliated by serum starvation and stained with antibodies against RABEP2 (red) and polyglutamylated tubulin (green). Knockdown of RABEP2 abolishes cilia formation. Knockdown of SDCCAG8 abolishes cilia formation and depletes RABEP2 localization from the centrioles in hTERT-RPE1 cells. Scale bars: 5 μ m. (E) RABEP2 localization at the centrosome is dependent on SDCCAG8. RABEP2- and SDCCAG8-positive pixels were quantitated at acetylated α-tubulin or polyglutamylated-tubulin positive centrosomes in siNS, siRABEP2 and siSDCCAG8 cells (C and D). While control cells (siNS) have 69.40 ± 1.194, n = 30, RABEP2-positive pixels at the centrosome, RABEP2-positive pixels are significantly reduced in siRABEP2 cells (13.00 ± 1.678, n = 30, ****p<0.0001) and in siSDCCAG8 cells (11.84 ± 1.231, n = 30, ****p<0.0001). Control cells (siNS) have 75.25 ± 2.145, n = 30 SDCCAG8-positive pixels at the centrosome, which is not changed in siRABEP2 cells (73.83 ± 2.138, n = 30), but is significantly reduced in siSDCCAG8 cells (10.76 ± 0.9907, n = 30, ****p<0.0001). Error bars, SEM; siNS, non-specific siRNA; siR2, RABEP2 siRNA; siS8, SDCCAG8 siRNA.

Article Snippet: Primary antibodies used were as follows: rabbit polyclonal anti-CEP164 was a gift from Erich Nigg (University of Basel, Switzerland), rabbit polyclonal anti-ERC1 (A302-698A, Bethyl Laboratories), rabbit polyclonal anti-CEP131 (ab99379, Abcam), rabbit polyclonal anti-RABEP2 (ab138279, Abcam), mouse monoclonal anti-SDCCAG8 (ab67098, Abcam), rabbit anti-DYKDDDDK Tag (#2368, Cell Signaling), mouse monoclonal anti-γ-tubulin (GTU-88, Sigma), rabbit anti-acetylated α-tubulin (#5335, Cell Signaling), mouse monoclonal anti-polyglutamylated tubulin (T9822, Sigma-Aldrich), rabbit polyclonal anti-CCDC41 (CEP83) (HPA038161, Atlas), rabbit polyclonal anti-FBF1 (HPA023677, Atlas), chicken anti-GFP (GFP-1020, Aves).

Techniques: Western Blot, Transfection, Staining

Journal: PLoS ONE

Article Title: SDCCAG8 Interacts with RAB Effector Proteins RABEP2 and ERC1 and Is Required for Hedgehog Signaling

doi: 10.1371/journal.pone.0156081

Figure Lengend Snippet: (A,B) hTERT-RPE1 cells were transfected with non-specific siRNA (siNS) and 24 hours later transfected with EGFP-PACT (A) or EGFP-RABEP2-PACT (B) constructs. Either of the tagged proteins localized to the centrosome and were detected with anti-GFP antibody (green). Ciliogenesis (acetylated-α-tubulin, red) was not affected in either of the situations. (C,D) hTERT-RPE1 cells were transfected with siSDCCAG8 and 24 hours later transfected with EGFP-PACT (C) or EGFP-RABEP2-PACT (D) constructs. Although, the tagged proteins localized to the centrosome (green), they failed to rescue the ciliogenesis defect (acetylated α-tubulin, red) in SDCCAG8 knockdown cells. Scale bars: 4 μ m. (E) Quantitation of the percentage of ciliated cells in (A, B, C and D), demonstrates that over expression of the EGFP-PACT or EGFP-RABEP2-PACT construct does not affect normal ciliation in siNS hTERT-RPE1 cells. Moreover, EGFP-RABEP2-PACT construct fails to rescue the ciliation defect in SDCCAG8 depleted cells (**p = 0.0068). Error bars, SEM. (F) We measured the length of cilia in EGFP-PACT and EGFP-RABEP2-PACT overexpression cells to examine whether it was affected. There was no significant difference in the length of cilia between siNS cells expressing EGFP-PACT and EGFP-RABEP2-PACT (73.08 ± 4.417 μ m, n = 3, vs. 67.80 ± 2.446 μ m, n = 3, p = 0.41). Similarly, the cilia length was uncorrected by the overexpression of EGFP-PACT and EGFP-RABEP2-PACT in siSDCCAG8 cells (13.36 ± 2.194 μ m, n = 3 vs. 14.58 ± 2.083 μ m, n = 3, p = 0.72). Error bars, SEM.

Article Snippet: Primary antibodies used were as follows: rabbit polyclonal anti-CEP164 was a gift from Erich Nigg (University of Basel, Switzerland), rabbit polyclonal anti-ERC1 (A302-698A, Bethyl Laboratories), rabbit polyclonal anti-CEP131 (ab99379, Abcam), rabbit polyclonal anti-RABEP2 (ab138279, Abcam), mouse monoclonal anti-SDCCAG8 (ab67098, Abcam), rabbit anti-DYKDDDDK Tag (#2368, Cell Signaling), mouse monoclonal anti-γ-tubulin (GTU-88, Sigma), rabbit anti-acetylated α-tubulin (#5335, Cell Signaling), mouse monoclonal anti-polyglutamylated tubulin (T9822, Sigma-Aldrich), rabbit polyclonal anti-CCDC41 (CEP83) (HPA038161, Atlas), rabbit polyclonal anti-FBF1 (HPA023677, Atlas), chicken anti-GFP (GFP-1020, Aves).

Techniques: Transfection, Construct, Quantitation Assay, Over Expression, Expressing

Journal: PLoS ONE

Article Title: SDCCAG8 Interacts with RAB Effector Proteins RABEP2 and ERC1 and Is Required for Hedgehog Signaling

doi: 10.1371/journal.pone.0156081

Figure Lengend Snippet: ( A , B ) Sdccag8 gt/gt cells have shorter cilia. Immunofluorescence staining of cultured mouse embryonic fibroblasts derived from wild type ( A ) and Sdccag8 gt/gt ( B ) mice using antibodies against distal appendage marker CEP164 (red) and cilia marker acetylated tubulin (green), demonstrate shorter cilia in Sdccag8 gt/gt cells ( B ). ( C ) Quantitation of the percentage of ciliated cells for wild type and Sdccag8 gt/gt cells after serum starvation (48 hrs). Significantly less Sdccag8 gt/gt cells grow cilia compared to wild type cells (18% vs. 93%, n = 100 for both genotypes, **p = 0.0018). ( D ) Sdccag8 gt/gt MEFs have significantly shorter cilia (1.9 ± 0.2 μm, n = 20) compared to wild type cells (2.9 ± 0.0 μm, n = 142, ****p<0.0001). ( E ) Sdccag8 gt/gt MEFs have attenuated response to Hh signal agonist SAG. qRT-PCR analysis demonstrates reduced levels of Hh pathway target gene Gli1 in SAG treated Sdccag8 gt/gt MEFs compared to wild type cells (N = 3). ( F ) Knockdown of SDCCAG8 causes a reduction in cilia formation in hTERT-RPE1 cells. Only 43% of SDCCAG8 knockdown cells grew cilia compared to 94% wild type cells (**p = 0.0092). ( G ) Cilia length is significantly reduced in SDCCAG8 knockdown cells (1.9 ± 0.1 μ, n = 63) compared to wild type cells (2.8 ± 0.1 μ., n = 80, ****p<0.0001). Scale bar: ( A , B ) 7.5 μm. wt , Sdccag8 wild type allele; gt , Sdccag8 gene-trap allele. ( H , I , J , K ) Immunofluorescence staining of control siNS hTERT-RPE1 cells ( H , J ) and siSDCCAG8 hTERT-RPE1 cells ( I , K ) with antibodies against acetylated tubulin (green) and distal appendage markers CEP83 (red) ( H , I ) or FBF1 (red) ( J , K ), reveals no abnormalities in the formation of centriolar distal appendages despite the loss of cilia in SDCCAG8 knockdown cells ( I , K ) compared to control cells transfected with non-specific siRNA ( F , H ).

Article Snippet: Primary antibodies used were as follows: rabbit polyclonal anti-CEP164 was a gift from Erich Nigg (University of Basel, Switzerland), rabbit polyclonal anti-ERC1 (A302-698A, Bethyl Laboratories), rabbit polyclonal anti-CEP131 (ab99379, Abcam), rabbit polyclonal anti-RABEP2 (ab138279, Abcam), mouse monoclonal anti-SDCCAG8 (ab67098, Abcam), rabbit anti-DYKDDDDK Tag (#2368, Cell Signaling), mouse monoclonal anti-γ-tubulin (GTU-88, Sigma), rabbit anti-acetylated α-tubulin (#5335, Cell Signaling), mouse monoclonal anti-polyglutamylated tubulin (T9822, Sigma-Aldrich), rabbit polyclonal anti-CCDC41 (CEP83) (HPA038161, Atlas), rabbit polyclonal anti-FBF1 (HPA023677, Atlas), chicken anti-GFP (GFP-1020, Aves).

Techniques: Immunofluorescence, Staining, Cell Culture, Derivative Assay, Marker, Quantitation Assay, Quantitative RT-PCR, Transfection

Journal: PLoS ONE

Article Title: SDCCAG8 Interacts with RAB Effector Proteins RABEP2 and ERC1 and Is Required for Hedgehog Signaling

doi: 10.1371/journal.pone.0156081

Figure Lengend Snippet: ( A ) Co-staining with the centriolar protein γ-tubulin demonstrates centrosomal localization for the identified SDCCAG8 interacting proteins RABEP2, ERC1, and CEP131 in hTERT-RPE1 cells. Note CEP131 localization also at centriolar satellites. Scale bars: 5 μm. ( B ) Co-staining with the ciliary axoneme marker poly-glutamylated tubulin (red) demonstrates ciliary and basal body localization of RABEP2, basal body localization of ERC1 and centriolar satellite localization of CEP131 in hTERT-RPE1 cells. Scale bars: 5 μm. ( C ) Quantification of the co-localization coefficients of RABEP2, ERC1 and CEP131 with γ-tubulin positive centrosomes in cells from ( A ). In each case n>40 centrosomes; error bars, SEM. ( D ) Quantification of the co-localization coefficients of RABEP2, ERC1 and CEP131 with polyglutamine-tubulin positive centrosomes in cells from ( B ). In each case n>40 centrosomes; error bars, SEM.

Article Snippet: Primary antibodies used were as follows: rabbit polyclonal anti-CEP164 was a gift from Erich Nigg (University of Basel, Switzerland), rabbit polyclonal anti-ERC1 (A302-698A, Bethyl Laboratories), rabbit polyclonal anti-CEP131 (ab99379, Abcam), rabbit polyclonal anti-RABEP2 (ab138279, Abcam), mouse monoclonal anti-SDCCAG8 (ab67098, Abcam), rabbit anti-DYKDDDDK Tag (#2368, Cell Signaling), mouse monoclonal anti-γ-tubulin (GTU-88, Sigma), rabbit anti-acetylated α-tubulin (#5335, Cell Signaling), mouse monoclonal anti-polyglutamylated tubulin (T9822, Sigma-Aldrich), rabbit polyclonal anti-CCDC41 (CEP83) (HPA038161, Atlas), rabbit polyclonal anti-FBF1 (HPA023677, Atlas), chicken anti-GFP (GFP-1020, Aves).

Techniques: Staining, Marker

Journal: PLoS ONE

Article Title: SDCCAG8 Interacts with RAB Effector Proteins RABEP2 and ERC1 and Is Required for Hedgehog Signaling

doi: 10.1371/journal.pone.0156081

Figure Lengend Snippet: (A) Western blot analysis of extracts from control or siRABEP2-depleted hTERT-RPE1 cells, probed with anti-RABEP2 or anti-β-actin, as a loading control. Dharmacon SMARTpool siRNAs were used to knock down endogenous RABEP2 at high efficiency. (B) RABEP2 knockdown abolishes cilia formation in hTERT-RPE1 cells. Control, RABEP2 and SDCCAG8 depleted hTERT-RPE1 cells were serum starved for 48 hours after transfection with siRNA, fixed, and immunostained for acetylated α-tubulin to label primary cilia. Percentage of ciliated cells was determined in each case, control 92.8 ± 5.2%, n = 50; siRABEP2 15 ± 8.6%, n = 50 and siSDCCAG8 31.48 ± 1.694%, n = 50. Error bars, SEM, ****p<0.0001. (C) hTERT-RPE1 cells were transfected with non-specific siRNA (siNS), siRABEP2, or siSDCCAG8. Cells were ciliated by serum starvation and stained with antibodies against SDCCAG8 (green) and acetylated α-tubulin (red). Knockdown of RABEP2 abolishes cilia formation, but does not alter SDCCAG8 localization at the centrioles. Knockdown of SDCCAG8 abolishes cilia formation in hTERT-RPE1 cells. Scale bars: 5 μ m. (D) hTERT-RPE1 cells were transfected with non-specific siRNA (siNS), siRABEP2, or siSDCCAG8. Cells were ciliated by serum starvation and stained with antibodies against RABEP2 (red) and polyglutamylated tubulin (green). Knockdown of RABEP2 abolishes cilia formation. Knockdown of SDCCAG8 abolishes cilia formation and depletes RABEP2 localization from the centrioles in hTERT-RPE1 cells. Scale bars: 5 μ m. (E) RABEP2 localization at the centrosome is dependent on SDCCAG8. RABEP2- and SDCCAG8-positive pixels were quantitated at acetylated α-tubulin or polyglutamylated-tubulin positive centrosomes in siNS, siRABEP2 and siSDCCAG8 cells (C and D). While control cells (siNS) have 69.40 ± 1.194, n = 30, RABEP2-positive pixels at the centrosome, RABEP2-positive pixels are significantly reduced in siRABEP2 cells (13.00 ± 1.678, n = 30, ****p<0.0001) and in siSDCCAG8 cells (11.84 ± 1.231, n = 30, ****p<0.0001). Control cells (siNS) have 75.25 ± 2.145, n = 30 SDCCAG8-positive pixels at the centrosome, which is not changed in siRABEP2 cells (73.83 ± 2.138, n = 30), but is significantly reduced in siSDCCAG8 cells (10.76 ± 0.9907, n = 30, ****p<0.0001). Error bars, SEM; siNS, non-specific siRNA; siR2, RABEP2 siRNA; siS8, SDCCAG8 siRNA.

Article Snippet: Primary antibodies used were as follows: rabbit polyclonal anti-CEP164 was a gift from Erich Nigg (University of Basel, Switzerland), rabbit polyclonal anti-ERC1 (A302-698A, Bethyl Laboratories), rabbit polyclonal anti-CEP131 (ab99379, Abcam), rabbit polyclonal anti-RABEP2 (ab138279, Abcam), mouse monoclonal anti-SDCCAG8 (ab67098, Abcam), rabbit anti-DYKDDDDK Tag (#2368, Cell Signaling), mouse monoclonal anti-γ-tubulin (GTU-88, Sigma), rabbit anti-acetylated α-tubulin (#5335, Cell Signaling), mouse monoclonal anti-polyglutamylated tubulin (T9822, Sigma-Aldrich), rabbit polyclonal anti-CCDC41 (CEP83) (HPA038161, Atlas), rabbit polyclonal anti-FBF1 (HPA023677, Atlas), chicken anti-GFP (GFP-1020, Aves).

Techniques: Western Blot, Transfection, Staining

Journal: PLoS ONE

Article Title: SDCCAG8 Interacts with RAB Effector Proteins RABEP2 and ERC1 and Is Required for Hedgehog Signaling

doi: 10.1371/journal.pone.0156081

Figure Lengend Snippet: (A,B) hTERT-RPE1 cells were transfected with non-specific siRNA (siNS) and 24 hours later transfected with EGFP-PACT (A) or EGFP-RABEP2-PACT (B) constructs. Either of the tagged proteins localized to the centrosome and were detected with anti-GFP antibody (green). Ciliogenesis (acetylated-α-tubulin, red) was not affected in either of the situations. (C,D) hTERT-RPE1 cells were transfected with siSDCCAG8 and 24 hours later transfected with EGFP-PACT (C) or EGFP-RABEP2-PACT (D) constructs. Although, the tagged proteins localized to the centrosome (green), they failed to rescue the ciliogenesis defect (acetylated α-tubulin, red) in SDCCAG8 knockdown cells. Scale bars: 4 μ m. (E) Quantitation of the percentage of ciliated cells in (A, B, C and D), demonstrates that over expression of the EGFP-PACT or EGFP-RABEP2-PACT construct does not affect normal ciliation in siNS hTERT-RPE1 cells. Moreover, EGFP-RABEP2-PACT construct fails to rescue the ciliation defect in SDCCAG8 depleted cells (**p = 0.0068). Error bars, SEM. (F) We measured the length of cilia in EGFP-PACT and EGFP-RABEP2-PACT overexpression cells to examine whether it was affected. There was no significant difference in the length of cilia between siNS cells expressing EGFP-PACT and EGFP-RABEP2-PACT (73.08 ± 4.417 μ m, n = 3, vs. 67.80 ± 2.446 μ m, n = 3, p = 0.41). Similarly, the cilia length was uncorrected by the overexpression of EGFP-PACT and EGFP-RABEP2-PACT in siSDCCAG8 cells (13.36 ± 2.194 μ m, n = 3 vs. 14.58 ± 2.083 μ m, n = 3, p = 0.72). Error bars, SEM.

Article Snippet: Primary antibodies used were as follows: rabbit polyclonal anti-CEP164 was a gift from Erich Nigg (University of Basel, Switzerland), rabbit polyclonal anti-ERC1 (A302-698A, Bethyl Laboratories), rabbit polyclonal anti-CEP131 (ab99379, Abcam), rabbit polyclonal anti-RABEP2 (ab138279, Abcam), mouse monoclonal anti-SDCCAG8 (ab67098, Abcam), rabbit anti-DYKDDDDK Tag (#2368, Cell Signaling), mouse monoclonal anti-γ-tubulin (GTU-88, Sigma), rabbit anti-acetylated α-tubulin (#5335, Cell Signaling), mouse monoclonal anti-polyglutamylated tubulin (T9822, Sigma-Aldrich), rabbit polyclonal anti-CCDC41 (CEP83) (HPA038161, Atlas), rabbit polyclonal anti-FBF1 (HPA023677, Atlas), chicken anti-GFP (GFP-1020, Aves).

Techniques: Transfection, Construct, Quantitation Assay, Over Expression, Expressing