Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: CCDC146 is required for sperm flagellum biogenesis and male fertility in mice

doi: 10.1007/s00018-023-05025-x

Figure Lengend Snippet: CCDC146 may facilitate ODF2 transportation by interacting with CCDC42 and CCDC38. A-C CCDC146 co-localized with γ-TUBULIN, CCDC38, and CCDC42 in NIH3T3 cells before serum starvation. pCSII-MYC-Ccdc146 and pEGFP-C1-Ccdc38 or pEGFP-C1-Ccdc42 were co-transfected into NIH3T3 cells. 24 h after transfection, cells were fixed and stained with anti-MYC and anti-GFP antibodies, and the nucleus was stained with DAPI. D CCDC42 cannot co-localize with Ac-TUBULIN in NIH3T3 cells during ciliogenesis. pEGFP-C1-Ccdc42 were transfected into NIH3T3 cells. After serum starvation, cells were fixed and stained with anti-GFP and anti-Ac-TUBULIN antibodies, and the nucleus was stained with DAPI. E CCDC38 co-localized with Ac-TUBULIN in NIH3T3 cells during ciliogenesis. pEGFP-C1-Ccdc38 were transfected into NIH3T3 cells. After serum starvation, cells were fixed and stained with anti-GFP and anti-Ac-TUBULIN antibodies, and the nucleus was stained with DAPI. F CCDC146 cannot co-localize with Ac-TUBULIN in NIH3T3 cells during ciliogenesis. pCSII-MYC-Ccdc146 were transfected into NIH3T3 cells. After serum starvation, cells were fixed and stained with anti-MYC and anti-Ac-TUBULIN antibodies, and the nucleus was stained with DAPI. G The immunofluorescence of CCDC146 in Ccdc146+/+ and Ccdc146−/− mice. Testis germ cells were stained with anti-CCDC146 antibody, and the nucleus was stained with DAPI. H The immunofluorescence of CCDC146 in Ccdc146+/+ and Ccdc146−/− mice. Spermatozoa from cauda of the epididymis were stained with anti-CCDC146 antibody, and the nucleus was stained with DAPI. Scale bars: 2·5 μm (A-F); 5 μm (G); 10 μm (A-F, H). I CCDC146 may interact with microtubule proteins and microtubule inner proteins. Co-IP of sperm flagellum proteins with FLAG-CCDC146 from testis lysate using anti-FLAG magnetic beads, followed by western blotting with anti-α-TUBULIN, anti-β-TUBULIN, anti-TEKT2, anti-SPACA9, anti-DYNLL2, anti-PPIL6, anti-NME5, anti-SPAG6, anti-SPAG16, and anti-FLAG (CCDC146) antibodies. MT microtubule; MIP microtubule inner protein; RS radial spoke; CPA central-pair apparatus

Article Snippet: The following primary antibodies were used for immunofluorescence (IF) and western blotting (WB): mouse anti-GFP (M20004, Abmart; 1:1000 for WB), rabbit anti-MYC (BE2011, EASYBIO; 1:1000 for WB), anti-ODF2 (12058-1-AP, Proteintech; 1:500 for WB, 1:200 for IF), anti-ODF1 (24736-1-AP, Proteintech; 1:1000 for WB), mouse anti-α-Tubulin antibody (AC012, ABclonal; 1:1000 for WB, 1:100 for IF), rabbit anti-β-Tubulin antibody (10068-1-AP, Proteintech; 1:1000 for WB), mouse anti-α/β-Tubulin antibody (ab44928, Abcam; 1:100 for IF), mouse anti-GAPDH antibody (AC002, ABclonal; 1:10,000 for WB), mouse anti-Ac-Tubulin antibody (T7451, Sigma-Aldrich; 1:200 for IF), mouse anti-NME5 antibody (12923-1-AP, Proteintech; 1:1000 for WB), rabbit anti-DYNLL2 antibody (16811-1-AP, Proteintech; 1:1000 for WB), rabbit anti-TEKT2 antibody (13518-1-AP, Proteintech; 1:1000 for WB), rabbit anti-SPACA9 antibody (26034-1-AP, Proteintech; 1:1000 for WB), rabbit anti-PPIL6 antibody (17452-1-AP, Proteintech; 1:1000 for WB), rabbit anti-NME5 antibody (12923-1-AP, Proteintech; 1:1000 for WB), mouse anti-SPAG6 antibody (H00008382-M04, Abnova; 1:1000 for WB), mouse anti-SPAG16 antibody (H00079582-M01, Abnova; 1:1000 for WB), rabbit anti-CCDC38 (generated by Dia-an Biotechnology, Wuhan, China; 1:500 for WB), rabbit anti-CCDC146 (generated by Dia-an Biotechnology, Wuhan, China; 1:200 for WB, 1:50 for IF).

Techniques: Transfection, Staining, Immunofluorescence, Co-Immunoprecipitation Assay, Magnetic Beads, Western Blot

Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: CCDC146 is required for sperm flagellum biogenesis and male fertility in mice

doi: 10.1007/s00018-023-05025-x

Figure Lengend Snippet: CCDC146 may facilitate ODF2 transportation by interacting with CCDC42 and CCDC38. A-C CCDC146 co-localized with γ-TUBULIN, CCDC38, and CCDC42 in NIH3T3 cells before serum starvation. pCSII-MYC-Ccdc146 and pEGFP-C1-Ccdc38 or pEGFP-C1-Ccdc42 were co-transfected into NIH3T3 cells. 24 h after transfection, cells were fixed and stained with anti-MYC and anti-GFP antibodies, and the nucleus was stained with DAPI. D CCDC42 cannot co-localize with Ac-TUBULIN in NIH3T3 cells during ciliogenesis. pEGFP-C1-Ccdc42 were transfected into NIH3T3 cells. After serum starvation, cells were fixed and stained with anti-GFP and anti-Ac-TUBULIN antibodies, and the nucleus was stained with DAPI. E CCDC38 co-localized with Ac-TUBULIN in NIH3T3 cells during ciliogenesis. pEGFP-C1-Ccdc38 were transfected into NIH3T3 cells. After serum starvation, cells were fixed and stained with anti-GFP and anti-Ac-TUBULIN antibodies, and the nucleus was stained with DAPI. F CCDC146 cannot co-localize with Ac-TUBULIN in NIH3T3 cells during ciliogenesis. pCSII-MYC-Ccdc146 were transfected into NIH3T3 cells. After serum starvation, cells were fixed and stained with anti-MYC and anti-Ac-TUBULIN antibodies, and the nucleus was stained with DAPI. G The immunofluorescence of CCDC146 in Ccdc146+/+ and Ccdc146−/− mice. Testis germ cells were stained with anti-CCDC146 antibody, and the nucleus was stained with DAPI. H The immunofluorescence of CCDC146 in Ccdc146+/+ and Ccdc146−/− mice. Spermatozoa from cauda of the epididymis were stained with anti-CCDC146 antibody, and the nucleus was stained with DAPI. Scale bars: 2·5 μm (A-F); 5 μm (G); 10 μm (A-F, H). I CCDC146 may interact with microtubule proteins and microtubule inner proteins. Co-IP of sperm flagellum proteins with FLAG-CCDC146 from testis lysate using anti-FLAG magnetic beads, followed by western blotting with anti-α-TUBULIN, anti-β-TUBULIN, anti-TEKT2, anti-SPACA9, anti-DYNLL2, anti-PPIL6, anti-NME5, anti-SPAG6, anti-SPAG16, and anti-FLAG (CCDC146) antibodies. MT microtubule; MIP microtubule inner protein; RS radial spoke; CPA central-pair apparatus

Article Snippet: Antibodies The following primary antibodies were used for immunofluorescence (IF) and western blotting (WB): mouse anti-GFP (M20004, Abmart; 1:1000 for WB), rabbit anti-MYC (BE2011, EASYBIO; 1:1000 for WB), anti-ODF2 (12058-1-AP, Proteintech; 1:500 for WB, 1:200 for IF), anti-ODF1 (24736-1-AP, Proteintech; 1:1000 for WB), mouse anti-α-Tubulin antibody (AC012, ABclonal; 1:1000 for WB, 1:100 for IF), rabbit anti-β-Tubulin antibody (10068-1-AP, Proteintech; 1:1000 for WB), mouse anti-α/β-Tubulin antibody (ab44928, Abcam; 1:100 for IF), mouse anti-GAPDH antibody (AC002, ABclonal; 1:10,000 for WB), mouse anti-Ac-Tubulin antibody (T7451, Sigma-Aldrich; 1:200 for IF), mouse anti-NME5 antibody (12923-1-AP, Proteintech; 1:1000 for WB), rabbit anti-DYNLL2 antibody (16811-1-AP, Proteintech; 1:1000 for WB), rabbit anti-TEKT2 antibody (13518-1-AP, Proteintech; 1:1000 for WB), rabbit anti-SPACA9 antibody (26034-1-AP, Proteintech; 1:1000 for WB), rabbit anti-PPIL6 antibody (17452-1-AP, Proteintech; 1:1000 for WB), rabbit anti-NME5 antibody (12923-1-AP, Proteintech; 1:1000 for WB), mouse anti-SPAG6 antibody (H00008382-M04, Abnova; 1:1000 for WB), mouse anti-SPAG16 antibody (H00079582-M01, Abnova; 1:1000 for WB), rabbit anti-CCDC38 (generated by Dia-an Biotechnology, Wuhan, China; 1:500 for WB), rabbit anti-CCDC146 (generated by Dia-an Biotechnology, Wuhan, China; 1:200 for WB, 1:50 for IF).

Techniques: Transfection, Staining, Immunofluorescence, Co-Immunoprecipitation Assay, Magnetic Beads, Western Blot