Journal: bioRxiv

Article Title: Tropism of SARS-CoV-2 for Developing Human Cortical Astrocytes

doi: 10.1101/2021.01.17.427024

Figure Lengend Snippet: A) Experimental paradigm for viral infection of human cortical tissue. Primary cortical tissue was acutely sectioned and cultured at the air-liquid interface. The next day tissue was infected with SARS-CoV-2 at MOI 0.5 and cultured for 72 hours before being collected and processed. B) SARS-CoV-2 infects GFAP+AQP4+ astrocyte cells in the developing human cortex, which are predominantly located in the subventricular zone (SVZ), where >81% of cells assayed expressed markers of astrocytes. 100% of infected cells expressed both SARS-CoV-2+ nucleocapsid (N) antibody and double-stranded (ds)RNA antibody. White arrowheads indicate dsRNA+GFAP+ infected astrocytes (dsRNA+SARS-CoV-2 N+ n=31 cells, GFAP+AQP4+ n=74 cells across two biological samples and four technical replicates). C) Few other neural types were infected, as indicated by co-labeling of SARS-CoV-2 N or dsRNA, where <8% of cells assayed expressed NEUN, a marker of cortical neurons, and <17% expressed KI67, a marker of dividing cells (NEUN n=613 cells, KI67 n=186 cells across two biological samples and four technical replicates). D) Vascular cell types can also be infected where 7% of LAMININ+ blood vessels, 13% of CD31+ endothelial cells and 18% of PDGFR-β+ mural cells have infection. White arrowheads indicate infected vascular cells (Laminin n=269 cells, CD31 n=247 cells, PDGFRB=225 cells across two biological samples and four technical replicates).

Article Snippet: Primary Antibodies include: Mouse: dsRNA, clone rJ2 (Millipore, MABE1134, 1:100), Sox2 (Santa Cruz, sc-365823, 1:500), S100B (Sigma, S2532, 1:200), Ki67 (Abcam, ab156956, 1:500), CD31 (Agilent, GA61061–2, 1:100), Olig2 (Millipore, MABN50, 1:100), Rabbit: SARS-CoV-2 (Sino Biological, 40143-R001, 1:200), Pax6 (Biolegend, 901301, 1:500), Hopx (Proteintech, 11419–1-AP, 1:500), Cleaved Caspase-3 (Cell Signaling, 9661S, 1:250), Synm (Proteintech, 20735–1-AP, 1:100), Aqp4 (Proteintech, 16473–1-AP, 1:600), Egfr (Abcam, ab32077, 1:100), Dpp4 (Proteintech, 10940–1-AP, 1:50), CD147 (Invitrogen, 34–5600, 1:500), Arcn1 (Proteintech, 23843–1-AP, 1:50), Rat: Gfap (Thermofisher, 13–0300, 1:200), Laminin (Abcam, ab80580, 1:500), Nrp1 (Abcam, ab81321, 1:50), Chicken: Gfap (Abcam, ab4674, 1:500), Map2 (Abcam, ab5392, 1:200), Goat: Ace2 (R&D, AF933, 1:200), Ace2 (Thermofisher, MA5–32307, 1:200), Iba1 (Abcam, ab48004, 1:500), Pdgfrb (R&D, AF385, 1:100), Sheep: Eomes (R&D, AF6166, 1:200), Guinea pig: NeuN (Millipore, ABN90, 1:500), Sheep: CD34 (R&D, AF7227, 1:200).

Techniques: Infection, Cell Culture, Labeling, Marker

Journal: bioRxiv

Article Title: Tropism of SARS-CoV-2 for Developing Human Cortical Astrocytes

doi: 10.1101/2021.01.17.427024

Figure Lengend Snippet: A) Viral infection of cortical organoids. Human stem cells from several lines were aggregated and differentiated toward cortical identity in suspension. After 5, 10, 16 or 22 weeks of differentiation, organoids were exposed to SARS-CoV-2 for 2 hours, media was replaced and then collected 72 hours later. B) After 5, 10, or 16 weeks organoids only indicated rare infection (white arrowheads). At five and 10 weeks, SARS-CoV-2 N+ cells did not co-express SOX2, NEUN or GFAP indicating infected cells are not cortical progenitors, neurons or astrocytes and may instead be an off-target population. However, after 16 weeks, in one stem cell line a few GFAP+ astrocytes were infected. C) Although rare cells are infected at neurogenic stages, as indicated by coronavirus N antibody presence, there was no observed viral replication with dsRNA at these timepoints. D) At late gliogenic stages, by week 22 infection was readily observed in GFAP astrocytes but not NEUN+ neurons. E) 94% of infected cells stained positive for markers of astrocytes GFAP or AQP4, while only 4% are NEUN+ neurons. White arrowheads indicate SARS-CoV-2+ dsRNA+ GFAP+ AQP4+ astrocytes (GFAP+AQP4+ n=169 cells, NEUN n=143 cells).

Article Snippet: Primary Antibodies include: Mouse: dsRNA, clone rJ2 (Millipore, MABE1134, 1:100), Sox2 (Santa Cruz, sc-365823, 1:500), S100B (Sigma, S2532, 1:200), Ki67 (Abcam, ab156956, 1:500), CD31 (Agilent, GA61061–2, 1:100), Olig2 (Millipore, MABN50, 1:100), Rabbit: SARS-CoV-2 (Sino Biological, 40143-R001, 1:200), Pax6 (Biolegend, 901301, 1:500), Hopx (Proteintech, 11419–1-AP, 1:500), Cleaved Caspase-3 (Cell Signaling, 9661S, 1:250), Synm (Proteintech, 20735–1-AP, 1:100), Aqp4 (Proteintech, 16473–1-AP, 1:600), Egfr (Abcam, ab32077, 1:100), Dpp4 (Proteintech, 10940–1-AP, 1:50), CD147 (Invitrogen, 34–5600, 1:500), Arcn1 (Proteintech, 23843–1-AP, 1:50), Rat: Gfap (Thermofisher, 13–0300, 1:200), Laminin (Abcam, ab80580, 1:500), Nrp1 (Abcam, ab81321, 1:50), Chicken: Gfap (Abcam, ab4674, 1:500), Map2 (Abcam, ab5392, 1:200), Goat: Ace2 (R&D, AF933, 1:200), Ace2 (Thermofisher, MA5–32307, 1:200), Iba1 (Abcam, ab48004, 1:500), Pdgfrb (R&D, AF385, 1:100), Sheep: Eomes (R&D, AF6166, 1:200), Guinea pig: NeuN (Millipore, ABN90, 1:500), Sheep: CD34 (R&D, AF7227, 1:200).

Techniques: Infection, Staining

Journal: Nature Communications

Article Title: Cep295 is a conserved scaffold protein required for generation of a bona fide mother centriole

doi: 10.1038/ncomms12567

Figure Lengend Snippet: ( a ) Centriolar distribution of Cep295 at different cell cycle stages. HeLa cells were stained with the indicated antibodies. The bottom panels represent quantification of the local signal intensity of centrin (left) and Cep295 (right). The local signal intensity was visualized in the indicated colours (M: mother centriole; D: daughter centriole). The schematic models represent the localization of Cep295, HsSAS-6 and centrin during the cell cycle. Note that HsSAS-6 disappears in late mitosis. In G1/S phase, the centrosomal linker connects the proximal end of two mother centrioles. Scale bar, 1 μm. ( b ) Cep295 localizes to the assembly site of procentrioles in the earliest stage (arrowheads) and forms a ring-like structure. The images were obtained by TCS SP8 HSR system using antibodies against Cep192 (green) and Cep295 (red). Scale bar, 500 nm. ( c ) 3D-SIM images representing top and side views of Cep295 at mother centrioles. GT335 was used as a centriole wall marker. An arrow points to recruitment of Cep295 onto the daughter centriole wall before the glutamylation of centriolar mitrotubules. Scale bar, 400 nm. ( d , e ) The graph shows the signal intensity of Cep295 at the mother centriole along the dotted line in ( c ). For quantification of the diameter, the distances between intensity maxima were measured (mean±s.d.).

Article Snippet: The following primary antibodies were used in this study: Rabbit polyclonal antibodies against Cep295/KIAA1731 (Sigma, HPA038596, IF 1:1000, WB 1:1000), Cep192 (a gift from Laurence Pelletier, IF 1:1000), Cep192 (Bethyl laboratories, A302–324A, WB 1:1000), Cep152 (Bethyl laboratories, A302–480A, IF 1:1000), Cep152 (Bethyl laboratories, A302–479A, WB 1:1000), CP110 (a gift from Brian David Dynlacht, IF 1:500), CP110 (proteintech, 12780–1-AP, IF 1:500), CPAP (a gift from Pierre Gönczy, IF 1:500), CPAP/CENP-J (Proteintech, 11517-1-AP, IF 1:500), STIL (Abcam, ab89314, IF 1:500, WB 1:1000), Cep135 (Abcam, ab196809, IF 1:1000); mouse monoclonal antibodies against centrin-2 (Millipore, 20H5, IF 1:1000), HsSAS-6 (Santa Cruz Bio-technology, Inc., sc-81431, IF 1:500, WB 1:1000), Plk4 (Merck Millipore, clone 6H5, MABC544, IF 1:500), γ-tubulin (GTU88) (Sigma-Aldrich, T5192, IF 1:1000), Polyglutamylation Modification (GT335, mAb) (AdipoGen, AG-20B-0020-C100, IF 1:5000), FLAG-tag (Sigma, F1804, IF 1:1000, WB 1:1000) and α-tubulin (Sigma-Aldrich, DM1A, IF1:1000); goat polyclonal antibody against GFP, FITC-conjugated (Abcam, ab6662, IF 1:300).

Techniques: Staining, Marker

Journal: Nature Communications

Article Title: Cep295 is a conserved scaffold protein required for generation of a bona fide mother centriole

doi: 10.1038/ncomms12567

Figure Lengend Snippet: ( a ) Schematic of Cep295 function described in this figure. ( b ) Three colour staining of centrioles in control and Cep295-depleted cells. The cells were stained with the indicated antibodies. ODF2 was used as an older mother centriole marker. Arrows point to a new mother centriole (M: older mother centriole; NM: new mother centriole). Scale bar, 1 μm. ( c , d ) HeLa cells transfected with control siRNA or Cep295 siRNA for 72 h were stained with indicated antibodies. CP110 and centrin were used as centriole markers . Scale bar, 1 μm. ( c ) The number shown in the bottom panels represents the number of Plk4/HsSAS-6/STIL foci. ( d ) Histograms represent frequency of interphase cells with ≧2 Plk4/HsSAS-6/STIL/CPAP foci in each condition. Values are mean percentages±s.e.m from three independent experiments ( N =30 for each condition). *** P <0.001; ** P <0.01, (two-tailed t -test). ( e ) HeLa cells were treated with control siRNA or Cep295 siRNA, followed by transfection with an empty vector (−) or, pCMV5-Plk4ΔPEST-FLAG wild-type. The cells were stained with the indicated antibodies. An arrow points to the defect in multiple centriole formation induced by PLK4 over-expression, upon Cep295 depletion. Scale bars, 5 μm in the low-magnified view, 1 μm in the inset. Schematic illustrations show frequency of interphase cells with 2 or 1 over-duplicated centriole foci. ( f ) Cep295-depleted HeLa cells were stained with the indicated antibodies. Almost all control cells harbour ⩾2 Cep192 and ⩾2 Cep152 foci per cell, whereas only 12 and 5% of Cep295-depleted cells have ⩾2 Cep192 and ⩾2 Cep152 foci per cell, respectively. Scale bar, 1 μm. ( g , h ) To monitor the expression levels of Cep295 at the mother and daughter centrioles, the triple staining analysis was performed with the indicated antibodies as shown in the representative panels. HeLa cells were transfected with control or Cep295 siRNA for 24 h. ( g ) Arrows point to the defective recruitment of PCM components in the Cep295-depleted cells just after disengagement. Scale bar, 1 μm. ( h ) Histograms represent frequency of late mitotic cells with the indicated category in each condition. Values are mean percentages±s.e.m from three independent experiments ( N =30 for each condition). ** P <0.01 (two-tailed t -test).

Article Snippet: The following primary antibodies were used in this study: Rabbit polyclonal antibodies against Cep295/KIAA1731 (Sigma, HPA038596, IF 1:1000, WB 1:1000), Cep192 (a gift from Laurence Pelletier, IF 1:1000), Cep192 (Bethyl laboratories, A302–324A, WB 1:1000), Cep152 (Bethyl laboratories, A302–480A, IF 1:1000), Cep152 (Bethyl laboratories, A302–479A, WB 1:1000), CP110 (a gift from Brian David Dynlacht, IF 1:500), CP110 (proteintech, 12780–1-AP, IF 1:500), CPAP (a gift from Pierre Gönczy, IF 1:500), CPAP/CENP-J (Proteintech, 11517-1-AP, IF 1:500), STIL (Abcam, ab89314, IF 1:500, WB 1:1000), Cep135 (Abcam, ab196809, IF 1:1000); mouse monoclonal antibodies against centrin-2 (Millipore, 20H5, IF 1:1000), HsSAS-6 (Santa Cruz Bio-technology, Inc., sc-81431, IF 1:500, WB 1:1000), Plk4 (Merck Millipore, clone 6H5, MABC544, IF 1:500), γ-tubulin (GTU88) (Sigma-Aldrich, T5192, IF 1:1000), Polyglutamylation Modification (GT335, mAb) (AdipoGen, AG-20B-0020-C100, IF 1:5000), FLAG-tag (Sigma, F1804, IF 1:1000, WB 1:1000) and α-tubulin (Sigma-Aldrich, DM1A, IF1:1000); goat polyclonal antibody against GFP, FITC-conjugated (Abcam, ab6662, IF 1:300).

Techniques: Staining, Control, Marker, Transfection, Two Tailed Test, Plasmid Preparation, Over Expression, Expressing

Journal: Nature Communications

Article Title: Cep295 is a conserved scaffold protein required for generation of a bona fide mother centriole

doi: 10.1038/ncomms12567

Figure Lengend Snippet: ( a ) 3D-SIM images showing centriolar distribution of Cep192 in control and Cep295-depleted HeLa cells (M: mother centriole; D: daughter centriole). Arrows point to the daughter centriole wall ( N =5). Dotted lines indicate the shape of centriole cylinders in this figure. Scale bar, 400 nm. ( b ) U2OS cells were transfected with Flag-tagged Cep192 plasmid for 24 h. The cells were fixed and stained with antibodies against Flag and Cep295 for STED microscope. The right panels show magnified views. Arrowhead points to the cap-like structure of Cep295 at the procentriole assembly site before recruitment of Cep192 ( N =10). Scale bar, 500 nm. The schematic illustrates the centriolar localization of Cep295 and Cep192. ( c ) STED images showing centriolar distribution of Cep295 in the presence or absence of HsSAS-6. HeLa cells were treated with control siRNA or HsSAS-6 siRNA for 48–60 h and stained with the indicated antibodies. To make sure the efficacy of HsSAS-6 depletion, we chose the Hs-SAS-6-depleted cells having only one mother centriole with a mono-polar spindle. Arrowhead shows centriolar distribution of Cep295 without the cartwheel structure ( N =3). Scale bar, 500 nm. ( d ) Control and Cep295-depleted HeLa cells were visualized by STED microscope using the indicated antibodies. Arrows point to poly-glutamylation of centriolar microtubules stained with GT335 antibody at the daughter centrioles ( N =3). Scale bar, 500 nm. ( e , f ) ( e ) HeLa cells treated with control siRNA or siRNA targeting endogenous Cep295 for 24 h were stained with antibodies against centrin1 (green) and Cep135 (red; a proximal component of centrioles). Nuclei are shown in blue. The arrow indicates defective recruitment of Cep135 to the disengaged daughter centriole. Scale bars, 5 μm in the low-magnified view, 1 μm in the inset. ( f ) Histograms represent frequency of late-mitotic cells with the indicated category in each condition. Values are mean percentages±s.e.m. from three independent experiments ( N =30 for each condition). ** P <0.01, (two-tailed t -test).

Article Snippet: The following primary antibodies were used in this study: Rabbit polyclonal antibodies against Cep295/KIAA1731 (Sigma, HPA038596, IF 1:1000, WB 1:1000), Cep192 (a gift from Laurence Pelletier, IF 1:1000), Cep192 (Bethyl laboratories, A302–324A, WB 1:1000), Cep152 (Bethyl laboratories, A302–480A, IF 1:1000), Cep152 (Bethyl laboratories, A302–479A, WB 1:1000), CP110 (a gift from Brian David Dynlacht, IF 1:500), CP110 (proteintech, 12780–1-AP, IF 1:500), CPAP (a gift from Pierre Gönczy, IF 1:500), CPAP/CENP-J (Proteintech, 11517-1-AP, IF 1:500), STIL (Abcam, ab89314, IF 1:500, WB 1:1000), Cep135 (Abcam, ab196809, IF 1:1000); mouse monoclonal antibodies against centrin-2 (Millipore, 20H5, IF 1:1000), HsSAS-6 (Santa Cruz Bio-technology, Inc., sc-81431, IF 1:500, WB 1:1000), Plk4 (Merck Millipore, clone 6H5, MABC544, IF 1:500), γ-tubulin (GTU88) (Sigma-Aldrich, T5192, IF 1:1000), Polyglutamylation Modification (GT335, mAb) (AdipoGen, AG-20B-0020-C100, IF 1:5000), FLAG-tag (Sigma, F1804, IF 1:1000, WB 1:1000) and α-tubulin (Sigma-Aldrich, DM1A, IF1:1000); goat polyclonal antibody against GFP, FITC-conjugated (Abcam, ab6662, IF 1:300).

Techniques: Control, Transfection, Plasmid Preparation, Staining, Microscopy, Two Tailed Test

Journal: Nature Communications

Article Title: Cep295 is a conserved scaffold protein required for generation of a bona fide mother centriole

doi: 10.1038/ncomms12567

Figure Lengend Snippet: ( a ) Physical interaction between endogenous Cep295 and Cep192. U2OS cells were transfected with Flag-tagged Cep192 full-length for 24 h. The proteins were then immunoprecipitated from cell lysates with Flag beads. Total cell lysate and immunoprecipitates (IPs) were analysed by western blotting using Flag, Cep295 or α-tubulin (loading control) antibodies. The same result was obtained in 293T cells. ( b ) Yeast two-hybrid assay showing the interaction between full-length Cep192 and full-length Cep295. The indicated clones were grown on the plates lacking histidine and containing 50 mM 3-AT at 30 °C. The same results were obtained from two independent clones for each combination. The outcome of the other clone is not shown. ( c ) Schematic of full-length Cep295 and the deletion mutants used for co-IP assays with Flag-tagged full-length Cep192 in U2OS cells. The sufficient and necessary regions for Cep192-binging in Cep295 are represented in pink and red, respectively. The DDC8-like domain, ALMS domain, and two evolutionarily conserved domains are indicated. ( d , e ) Co-immunoprecipitation assay in U2OS cells testing interaction between Flag-Cep192 and the indicated Cep295-GFP deletion mutants or between Cep295 fragment containing the Cep192-interacting region and N-terminal or C-terminal fragment of Cep192. The Flag-tagged proteins were immunoprecipitated using Flag beads from the cell lysate. Total cell lysates and IPs were analysed by western blotting using the indicated antibodies. ( f ) Three colour staining of Cep295-depleted HeLa cells expressing full-length Cep295 and the mutant protein lacking aa 1942–2431. The phenotype was analysed mostly in the first round of cell cycle (∼48 h after the RNAi treatment). The cells were analysed by TCS SP8 HSR system using antibodies against Cep192 (green), Cep295 (red) and CP110 (blue). Arrows point to the daughter centriole wall. M: mother centriole indicated as a cylinder. Scale bar, 1 μm. ( g ) For rescue experiments, Cep295-depleted HeLa cells were transfected with full-length Cep295 and the indicated mutant. Histograms represent frequency of interphase cells with the indicated number of γ-tubulin foci in each condition. Values are mean percentages±s.e.m from three independent experiments ( N >50 for each condition). * P <0.05 (two-tailed t -test).

Article Snippet: The following primary antibodies were used in this study: Rabbit polyclonal antibodies against Cep295/KIAA1731 (Sigma, HPA038596, IF 1:1000, WB 1:1000), Cep192 (a gift from Laurence Pelletier, IF 1:1000), Cep192 (Bethyl laboratories, A302–324A, WB 1:1000), Cep152 (Bethyl laboratories, A302–480A, IF 1:1000), Cep152 (Bethyl laboratories, A302–479A, WB 1:1000), CP110 (a gift from Brian David Dynlacht, IF 1:500), CP110 (proteintech, 12780–1-AP, IF 1:500), CPAP (a gift from Pierre Gönczy, IF 1:500), CPAP/CENP-J (Proteintech, 11517-1-AP, IF 1:500), STIL (Abcam, ab89314, IF 1:500, WB 1:1000), Cep135 (Abcam, ab196809, IF 1:1000); mouse monoclonal antibodies against centrin-2 (Millipore, 20H5, IF 1:1000), HsSAS-6 (Santa Cruz Bio-technology, Inc., sc-81431, IF 1:500, WB 1:1000), Plk4 (Merck Millipore, clone 6H5, MABC544, IF 1:500), γ-tubulin (GTU88) (Sigma-Aldrich, T5192, IF 1:1000), Polyglutamylation Modification (GT335, mAb) (AdipoGen, AG-20B-0020-C100, IF 1:5000), FLAG-tag (Sigma, F1804, IF 1:1000, WB 1:1000) and α-tubulin (Sigma-Aldrich, DM1A, IF1:1000); goat polyclonal antibody against GFP, FITC-conjugated (Abcam, ab6662, IF 1:300).

Techniques: Transfection, Immunoprecipitation, Western Blot, Control, Y2H Assay, Clone Assay, Co-Immunoprecipitation Assay, Staining, Expressing, Mutagenesis, Two Tailed Test

Journal: Nature Communications

Article Title: Cep295 is a conserved scaffold protein required for generation of a bona fide mother centriole

doi: 10.1038/ncomms12567

Figure Lengend Snippet: ( a ) Schematic of full-length Cep192 and the deletion mutants used for co-IP assays with endogenous Cep295 in U2OS cells. The minimal Cep295-binging region in Cep192 is represented in grey. ( b ) U2OS cells were transfected with Flag-Cep192 full-length and the indicated mutants. The cells were stained with antibodies against Cep192 (green) and CP110 (red). Scale bar, 1 μm. ( c , d ) U2OS cells expressing Flag-Cep192 full length or the deletion mutant proteins were immunoprecipitated with FLAG beads. Total cell lysates and IPs were analysed by western blotting using Cep295, Flag or tubulin antibodies. ( e ) GST pull-down assay showing the interaction between Cep295 and Cep192 fragments (aa 1727–2204 of Cep295 and aa 1501–2040 of Cep192) in vitro. These bacterially purified recombinant proteins contain the interacting regions that were identified by co-immunoprecipitation experiments. ( f , g ) For rescue experiments, Cep192-depleted U2OS cells were transfected with RNAi-resistant full-length Cep295 and the indicated mutant. Scale bar, 5 μm. Histograms represent frequency of bipolar mitotic spindles with the indicated number of γ-tubulin foci. Values are mean percentages±s.e.m from three independent experiments ( N =30 for each condition). * P <0.05 (two-tailed t -test). ( h , i ) Dominant negative effects of the Cep192 fragment that binds to Cep295. U2OS cells were transfected with full-length Cep295 and the indicated mutant. Scale bar, 1 μm. Histograms represent frequency of transfected cells with the indicated intensity of γ-tubulin foci. Values are mean percentages±s.e.m from three independent experiments ( N =30 for each condition). ** P <0.01 (two-tailed t -test).

Article Snippet: The following primary antibodies were used in this study: Rabbit polyclonal antibodies against Cep295/KIAA1731 (Sigma, HPA038596, IF 1:1000, WB 1:1000), Cep192 (a gift from Laurence Pelletier, IF 1:1000), Cep192 (Bethyl laboratories, A302–324A, WB 1:1000), Cep152 (Bethyl laboratories, A302–480A, IF 1:1000), Cep152 (Bethyl laboratories, A302–479A, WB 1:1000), CP110 (a gift from Brian David Dynlacht, IF 1:500), CP110 (proteintech, 12780–1-AP, IF 1:500), CPAP (a gift from Pierre Gönczy, IF 1:500), CPAP/CENP-J (Proteintech, 11517-1-AP, IF 1:500), STIL (Abcam, ab89314, IF 1:500, WB 1:1000), Cep135 (Abcam, ab196809, IF 1:1000); mouse monoclonal antibodies against centrin-2 (Millipore, 20H5, IF 1:1000), HsSAS-6 (Santa Cruz Bio-technology, Inc., sc-81431, IF 1:500, WB 1:1000), Plk4 (Merck Millipore, clone 6H5, MABC544, IF 1:500), γ-tubulin (GTU88) (Sigma-Aldrich, T5192, IF 1:1000), Polyglutamylation Modification (GT335, mAb) (AdipoGen, AG-20B-0020-C100, IF 1:5000), FLAG-tag (Sigma, F1804, IF 1:1000, WB 1:1000) and α-tubulin (Sigma-Aldrich, DM1A, IF1:1000); goat polyclonal antibody against GFP, FITC-conjugated (Abcam, ab6662, IF 1:300).

Techniques: Co-Immunoprecipitation Assay, Transfection, Staining, Expressing, Mutagenesis, Immunoprecipitation, Western Blot, Pull Down Assay, In Vitro, Purification, Recombinant, Two Tailed Test, Dominant Negative Mutation

Journal: Nature Communications

Article Title: Cep295 is a conserved scaffold protein required for generation of a bona fide mother centriole

doi: 10.1038/ncomms12567

Figure Lengend Snippet: A speculative model of the role of Cep295 in centriole biogenesis. Cep295 promotes recruitment of Cep192 onto the wall of a newly formed daughter centriole. Cep295 is also critical for the integrity of the proximal part of the daughter centriole and for stability of the resulting centriole in the next cell cycle. The events coordinated by Cep295 are thus critical for the ability of a new mother centriole to duplicate, recruit PCM components and act as the MTOC.

Article Snippet: The following primary antibodies were used in this study: Rabbit polyclonal antibodies against Cep295/KIAA1731 (Sigma, HPA038596, IF 1:1000, WB 1:1000), Cep192 (a gift from Laurence Pelletier, IF 1:1000), Cep192 (Bethyl laboratories, A302–324A, WB 1:1000), Cep152 (Bethyl laboratories, A302–480A, IF 1:1000), Cep152 (Bethyl laboratories, A302–479A, WB 1:1000), CP110 (a gift from Brian David Dynlacht, IF 1:500), CP110 (proteintech, 12780–1-AP, IF 1:500), CPAP (a gift from Pierre Gönczy, IF 1:500), CPAP/CENP-J (Proteintech, 11517-1-AP, IF 1:500), STIL (Abcam, ab89314, IF 1:500, WB 1:1000), Cep135 (Abcam, ab196809, IF 1:1000); mouse monoclonal antibodies against centrin-2 (Millipore, 20H5, IF 1:1000), HsSAS-6 (Santa Cruz Bio-technology, Inc., sc-81431, IF 1:500, WB 1:1000), Plk4 (Merck Millipore, clone 6H5, MABC544, IF 1:500), γ-tubulin (GTU88) (Sigma-Aldrich, T5192, IF 1:1000), Polyglutamylation Modification (GT335, mAb) (AdipoGen, AG-20B-0020-C100, IF 1:5000), FLAG-tag (Sigma, F1804, IF 1:1000, WB 1:1000) and α-tubulin (Sigma-Aldrich, DM1A, IF1:1000); goat polyclonal antibody against GFP, FITC-conjugated (Abcam, ab6662, IF 1:300).

Techniques: