Journal: eLife

Article Title: Lack of CCDC146, a ubiquitous centriole and microtubule-associated protein, leads to non-syndromic male infertility in human and mouse

doi: 10.7554/eLife.86845

Figure Lengend Snippet: ( A ) Mouse epididymal spermatozoa observed with conventional IF. WT and HA-CCDC146 sperm were labeled with anti-HA Tag Alexa Fluor 488-conjugated 3 (green) Abs (anti-HA #1). DNA was stained with Hoechst (blue). The upper image shows the staining of a WT sperm and the lower images, the staining of a HA-CCDC146 sperm. A punctuate signal is observed in HA-CCDC146 sperm (scale bars 5 µm). ( B ) Quantification of the density of dots per square µm (µm 2 ) and statistical significance between WT and HA-CCDC146 sperm was assessed by Mann–Whitney test (n=58 for WT and 65 for HA_CCDC146), p-value as indicated.p-value of 0.05 or lower was considered statistically significant. Error bars show standard deviation. ( C ) Mouse epididymal spermatozoa observed with expansion microscopy. HA-CCDC146 sperm were immunolabeled with anti-β-tubulin (red) and anti-HA #2 (green) Abs, and DNA was stained with Hoechst (blue). The right image shows the sperm with merged immunostaining. Scale bars 5 µm. ( D ) Mouse epididymal spermatozoa observed with expansion microscopy. The lower images show the staining, HA-CCDC146 (red), tubulin (green), and merge observed in the principal piece of the flagellum. Strong red punctiform signals were observed at the level of axonemal breakages induced by the expansion process. White arrows indicate the zones of the micro breaks. Scale bars 10 μm.

Article Snippet: HA Tag Alexa Fluor–488-conjugated Antibody , , Cell Signaling , 28427 , IF:1/400.

Techniques: Labeling, Staining, MANN-WHITNEY, Standard Deviation, Microscopy, Immunolabeling, Immunostaining

Journal: eLife

Article Title: Lack of CCDC146, a ubiquitous centriole and microtubule-associated protein, leads to non-syndromic male infertility in human and mouse

doi: 10.7554/eLife.86845

Figure Lengend Snippet: ( A ) Western blot of WT and HA-CCDC146 sperm extract solubilized with N-lauroylsarcosine (sarkosyl), an anionic detergent. Sarkosyl was used at increasing concentrations (0.2 and 0.4%). The presence of HA-CCDC146 was detected by an anti-HA Ab. ( B ) Western blot of WT and HA-CCDC146 sperm extracts solubilized with alternative detergents (RIPA, CHAPS, Tris-HCl) and whole sperm extract solubilized in Laemmli. The presence of HA-CCDC146 was revealed by an anti-HA Ab. ( C ) Epididymal HA-CCDC146 sperm ( C1–C2 ) and WT sperm ( C3–C4 ), treated with sarkosyl (5 min, 0.2% sarkosyl) or not (NT), were immunostained to reveal the HA-tag (red) and counterstained with Hoechst (blue). ( C1 ) Without treatment, a faint CCDC146 signal (white arrowheads) is observed along the flagellum from HA-CCDC146 sperm. ( C2 ) Treatment with sarkosyl enhanced the HA-CCDC146 signal along the sperm flagellum. ( C3 ) The HA signal present the midpiece is likely non-specific since it is present in WT non-treated (NT) sperm. See also , suggesting that this signal is due to secondary Abs. ( C4 ) The HA signal in WT sperm is not enhanced by sarkosyl treatment. Scale bars 10 μm. Figure 10—source data 1. Uncropped gel of : tagged sperm. Figure 10—source data 2. Uncropped gel of : WT sperm. Figure 10—source data 3. Uncropped gel of .

Article Snippet: HA Tag Alexa Fluor–488-conjugated Antibody , , Cell Signaling , 28427 , IF:1/400.

Techniques: Western Blot

Journal: eLife

Article Title: Lack of CCDC146, a ubiquitous centriole and microtubule-associated protein, leads to non-syndromic male infertility in human and mouse

doi: 10.7554/eLife.86845

Figure Lengend Snippet:

Article Snippet: HA Tag Alexa Fluor–488-conjugated Antibody , , Cell Signaling , 28427 , IF:1/400.

Techniques: Transfection, Construct, Plasmid Preparation, Recombinant, Sequencing, TUNEL Assay

Journal: eLife

Article Title: Lack of CCDC146, a ubiquitous centriole and microtubule-associated protein, leads to non-syndromic male infertility in human and mouse

doi: 10.7554/eLife.86845

Figure Lengend Snippet:

Article Snippet: HA Tag Alexa Fluor–488-conjugated Antibody , , Cell Signaling , 28427 , IF:1/400.

Techniques: Produced