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    Abmart Inc mouse anti gfp
    The knockout of Ccdc146 leads to male infertility. A CCDC146 was related to CCDC38 predicted by the STRING database. B CCDC146 interacted with CCDC38. pCSII-MYC-Ccdc146 was transfected into HEK293T cells with pEGFP-C1-Ccdc38, 48 hours after transfection, cells were collected for immunoprecipitation with anti-MYC, and detected <t>by</t> <t>anti-GFP</t> or anti-MYC antibodies, respectively. C CCDC146 co-localized with CCDC38 and γ-TUBULIN in Hela cells. pCSII-MYC-Ccdc146 and pEGFP-C1-Ccdc38 were co-transfected into Hela cells. 24 h after transfection, cells were fixed and stained with anti-MYC and anti-γ-TUBULIN <t>or</t> <t>anti-GFP</t> antibodies, and the nucleus was stained with DAPI. Scale bar: 5 μm. D The expression of Ccdc146 in different tissues of adult mice. GAPDH was used as a loading control. E The expression of Ccdc146 on different days. TUBULIN was used as a loading control. F The schematic illustration of the knockout strategy for generating Ccdc146−/− mice lacking exon 3–7. Primer-F1, Primer-R1, and Primer-R2 were used as the genotyping primers. G Genotyping of Ccdc146−/− mice. H Western blotting of CCDC146 indicated the depletion efficiency in Ccdc146−/− male mice. I Ccdc146−/− male mice were infertile. The Y axis represents the average number of offspring per litter. Data are presented as means ± SEM. two-tailed Student’s t test; ***P < 0·001. J The number of pups per litter of the different crossing possibilities. Data are presented as means ± SEM. two-tailed Student’s t test; ns: no significance; ***P < 0.001. K The sex ratio of the pups of the different crossing possibilities. Data are presented as means ± SEM. two-tailed Student’s t test; ns: no significance; ***P < 0.001. L The size of the Ccdc146+/+ and Ccdc146−/− mice testes were unaffected. M Testis from Ccdc146+/+ and Ccdc146−/− male mice had no obvious difference in weight (n = 5). Data are presented as means ± SEM. two-tailed Student’s t-test; ns: no significance. N The body weight of Ccdc146+/+ and Ccdc146−/− male mice had no obvious difference (n = 5). Data are presented as means ± SEM. two-tailed Student’s t-test; ns: no significance. O The testis/body weight ratios in Ccdc146+/+ and Ccdc146−/− male mice were consistent (n = 5). Data are presented as means ± SEM. two-tailed Student’s t-test; ns no significance
    Mouse Anti Gfp, supplied by Abmart Inc, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/mouse anti gfp/product/Abmart Inc
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    1) Product Images from "CCDC146 is required for sperm flagellum biogenesis and male fertility in mice"

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

    Journal: Cellular and Molecular Life Sciences: CMLS

    doi: 10.1007/s00018-023-05025-x

    The knockout of Ccdc146 leads to male infertility. A CCDC146 was related to CCDC38 predicted by the STRING database. B CCDC146 interacted with CCDC38. pCSII-MYC-Ccdc146 was transfected into HEK293T cells with pEGFP-C1-Ccdc38, 48 hours after transfection, cells were collected for immunoprecipitation with anti-MYC, and detected by anti-GFP or anti-MYC antibodies, respectively. C CCDC146 co-localized with CCDC38 and γ-TUBULIN in Hela cells. pCSII-MYC-Ccdc146 and pEGFP-C1-Ccdc38 were co-transfected into Hela cells. 24 h after transfection, cells were fixed and stained with anti-MYC and anti-γ-TUBULIN or anti-GFP antibodies, and the nucleus was stained with DAPI. Scale bar: 5 μm. D The expression of Ccdc146 in different tissues of adult mice. GAPDH was used as a loading control. E The expression of Ccdc146 on different days. TUBULIN was used as a loading control. F The schematic illustration of the knockout strategy for generating Ccdc146−/− mice lacking exon 3–7. Primer-F1, Primer-R1, and Primer-R2 were used as the genotyping primers. G Genotyping of Ccdc146−/− mice. H Western blotting of CCDC146 indicated the depletion efficiency in Ccdc146−/− male mice. I Ccdc146−/− male mice were infertile. The Y axis represents the average number of offspring per litter. Data are presented as means ± SEM. two-tailed Student’s t test; ***P < 0·001. J The number of pups per litter of the different crossing possibilities. Data are presented as means ± SEM. two-tailed Student’s t test; ns: no significance; ***P < 0.001. K The sex ratio of the pups of the different crossing possibilities. Data are presented as means ± SEM. two-tailed Student’s t test; ns: no significance; ***P < 0.001. L The size of the Ccdc146+/+ and Ccdc146−/− mice testes were unaffected. M Testis from Ccdc146+/+ and Ccdc146−/− male mice had no obvious difference in weight (n = 5). Data are presented as means ± SEM. two-tailed Student’s t-test; ns: no significance. N The body weight of Ccdc146+/+ and Ccdc146−/− male mice had no obvious difference (n = 5). Data are presented as means ± SEM. two-tailed Student’s t-test; ns: no significance. O The testis/body weight ratios in Ccdc146+/+ and Ccdc146−/− male mice were consistent (n = 5). Data are presented as means ± SEM. two-tailed Student’s t-test; ns no significance
    Figure Legend Snippet: The knockout of Ccdc146 leads to male infertility. A CCDC146 was related to CCDC38 predicted by the STRING database. B CCDC146 interacted with CCDC38. pCSII-MYC-Ccdc146 was transfected into HEK293T cells with pEGFP-C1-Ccdc38, 48 hours after transfection, cells were collected for immunoprecipitation with anti-MYC, and detected by anti-GFP or anti-MYC antibodies, respectively. C CCDC146 co-localized with CCDC38 and γ-TUBULIN in Hela cells. pCSII-MYC-Ccdc146 and pEGFP-C1-Ccdc38 were co-transfected into Hela cells. 24 h after transfection, cells were fixed and stained with anti-MYC and anti-γ-TUBULIN or anti-GFP antibodies, and the nucleus was stained with DAPI. Scale bar: 5 μm. D The expression of Ccdc146 in different tissues of adult mice. GAPDH was used as a loading control. E The expression of Ccdc146 on different days. TUBULIN was used as a loading control. F The schematic illustration of the knockout strategy for generating Ccdc146−/− mice lacking exon 3–7. Primer-F1, Primer-R1, and Primer-R2 were used as the genotyping primers. G Genotyping of Ccdc146−/− mice. H Western blotting of CCDC146 indicated the depletion efficiency in Ccdc146−/− male mice. I Ccdc146−/− male mice were infertile. The Y axis represents the average number of offspring per litter. Data are presented as means ± SEM. two-tailed Student’s t test; ***P < 0·001. J The number of pups per litter of the different crossing possibilities. Data are presented as means ± SEM. two-tailed Student’s t test; ns: no significance; ***P < 0.001. K The sex ratio of the pups of the different crossing possibilities. Data are presented as means ± SEM. two-tailed Student’s t test; ns: no significance; ***P < 0.001. L The size of the Ccdc146+/+ and Ccdc146−/− mice testes were unaffected. M Testis from Ccdc146+/+ and Ccdc146−/− male mice had no obvious difference in weight (n = 5). Data are presented as means ± SEM. two-tailed Student’s t-test; ns: no significance. N The body weight of Ccdc146+/+ and Ccdc146−/− male mice had no obvious difference (n = 5). Data are presented as means ± SEM. two-tailed Student’s t-test; ns: no significance. O The testis/body weight ratios in Ccdc146+/+ and Ccdc146−/− male mice were consistent (n = 5). Data are presented as means ± SEM. two-tailed Student’s t-test; ns no significance

    Techniques Used: Knock-Out, Transfection, Immunoprecipitation, Staining, Expressing, Western Blot, Two Tailed Test

    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
    Figure Legend 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

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

    CCDC146 interacts with CCDC38 and CCDC42. A CCDC146 interacted with CCDC42. pCSII-MYC-Ccdc146 were transfected into HEK293T cells with pEGFP-C1-Ccdc42 48 hours after transfection; cells were collected for immunoprecipitation with anti-MYC, and detected by anti-GFP or anti-MYC antibodies, respectively. B CCDC146 interacted with CCDC38 and CCDC42 in testis. Co-IP of CCDC38 and CCDC42 with FLAG-CCDC146 from testis lysate using anti-FLAG magnetic beads, followed by western blotting with anti-CCDC38, anti-CCDC42, anti-CCDC146 and anti-FLAG (CCDC146) antibodies. C, D Western blots showing CCDC38, CCDC42, ODF2, IFT88, IFT20, and ODF1 protein levels in lysates from Ccdc146+/+ and Ccdc146−/− mice testes. GAPDH served as a loading control. ODF2, IFT88, and IFT20 protein levels were reduced in Ccdc146−/− testes compared with Ccdc146+/+ testes. Data are presented as means ± SEM. two-tailed Student’s t test; ns: no significance; *P < 0·1; **P < 0·01. E Binding mode of CCDC38 on the CCDC146 predicted by docking. Detailed interaction network between CCDC38 and CCDC146. Key residues of CCDC146 (blue) and CCDC38 (light red) are displayed as sticks. H-bonds are displayed in dash lines. F Binding mode of CCDC42 on the CCDC146 predicted by docking. Detailed interaction network between CCDC42 and CCDC146. Key residues of CCDC146 (blue) and CCDC38 (light yellow) are displayed as sticks. H-bonds are displayed in yellow dashed lines. G Alignment of the mode of CCDC38 and CCDC42 on the CCDC146 predicted by docking. H–I Interactions between CCDC38 and WT or D541, or R547 mutants of truncated CCDC146 were detected by Co-IP assays. J–K Co-IP assays detected interactions between CCDC42 and WT or L232, or Y245 mutants of truncated CCDC146
    Figure Legend Snippet: CCDC146 interacts with CCDC38 and CCDC42. A CCDC146 interacted with CCDC42. pCSII-MYC-Ccdc146 were transfected into HEK293T cells with pEGFP-C1-Ccdc42 48 hours after transfection; cells were collected for immunoprecipitation with anti-MYC, and detected by anti-GFP or anti-MYC antibodies, respectively. B CCDC146 interacted with CCDC38 and CCDC42 in testis. Co-IP of CCDC38 and CCDC42 with FLAG-CCDC146 from testis lysate using anti-FLAG magnetic beads, followed by western blotting with anti-CCDC38, anti-CCDC42, anti-CCDC146 and anti-FLAG (CCDC146) antibodies. C, D Western blots showing CCDC38, CCDC42, ODF2, IFT88, IFT20, and ODF1 protein levels in lysates from Ccdc146+/+ and Ccdc146−/− mice testes. GAPDH served as a loading control. ODF2, IFT88, and IFT20 protein levels were reduced in Ccdc146−/− testes compared with Ccdc146+/+ testes. Data are presented as means ± SEM. two-tailed Student’s t test; ns: no significance; *P < 0·1; **P < 0·01. E Binding mode of CCDC38 on the CCDC146 predicted by docking. Detailed interaction network between CCDC38 and CCDC146. Key residues of CCDC146 (blue) and CCDC38 (light red) are displayed as sticks. H-bonds are displayed in dash lines. F Binding mode of CCDC42 on the CCDC146 predicted by docking. Detailed interaction network between CCDC42 and CCDC146. Key residues of CCDC146 (blue) and CCDC38 (light yellow) are displayed as sticks. H-bonds are displayed in yellow dashed lines. G Alignment of the mode of CCDC38 and CCDC42 on the CCDC146 predicted by docking. H–I Interactions between CCDC38 and WT or D541, or R547 mutants of truncated CCDC146 were detected by Co-IP assays. J–K Co-IP assays detected interactions between CCDC42 and WT or L232, or Y245 mutants of truncated CCDC146

    Techniques Used: Transfection, Immunoprecipitation, Co-Immunoprecipitation Assay, Magnetic Beads, Western Blot, Two Tailed Test, Binding Assay



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    Large TMEM24-positive ER/PM junctions at cell–cell interfaces. (A) Left: Spinning disk confocal images of TMEM24-mCherry, C2CD2-eGFP, and JPH4-eGFP in HEK293 cells. Center: <t>plasma</t> <t>membranes</t> labeled with CellBrite 650 dye. Faint diagonal lines have been added to indicate regions of the micrographs occupied by the cells. Right: ER/PM junctions from the left fields with cell-adjacent ER/PM junctions are outlined in red. (B) ROIs were drawn around regions of the plasma membrane touching an adjacent cell or facing open space and the mean fluorescence intensity of the indicated proteins within those regions was measured. An enrichment ratio was calculated for each cell by dividing the mean fluorescent intensity at adjacent regions by the mean non-adjacent fluorescent intensities. Student’s t test; P values of P = 0.0010 for TMEM24 and P = 7.12902E-07 for C2CD2. (C and D) Cells expressing TMEM24-eGFP and TMEM24-mCherry were coplated as illustrated by the diagram. (C) TMEM24 expressed in adjacent cells forms symmetrical ER/PM junctions. (D) Gaps in the TMEM24 signal (indicated by arrowheads) in one cell are mirrored by gaps in the signal of the adjacent cell. (E) Diagram illustrating TMEM24 behavior in adjacent cells expressing TMEM24. (F) TMEM24 tagged with <t>GFP</t> at the endogenous locus in IMR32 cells localizes preferentially to the cell–cell interface. (G) Quantification of the enrichment ratio for mean fluorescence of TMEM24 tagged at the endogenous locus (endoTMEM24) at regions of IMR32 cells that face adjacent cells or empty space in the dish. As endoTMEM24 signal may stem from both adjacent cells, the observed fluorescence at the adjacent region has been halved and compared against non-adjacent regions (P = 0.000000003, n = 24 cells). (H) Differentiated IMR32 cells show that endoTMEM24 fluorescence is preferentially localized at sites of cell–cell contact as in the undifferentiated IMR32 cells. In F and H, the plasma membrane was labeled with CellBrite 650 dye. Diagonal lines indicate regions of the micrographs occupied by cells to clearly differentiate these regions from empty spaces. Scale bars = 5 μm.
    Mouse Anti Gfp, supplied by Roche, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    mouse monoclonal anti gfp  (Roche)
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    Large TMEM24-positive ER/PM junctions at cell–cell interfaces. (A) Left: Spinning disk confocal images of TMEM24-mCherry, C2CD2-eGFP, and JPH4-eGFP in HEK293 cells. Center: <t>plasma</t> <t>membranes</t> labeled with CellBrite 650 dye. Faint diagonal lines have been added to indicate regions of the micrographs occupied by the cells. Right: ER/PM junctions from the left fields with cell-adjacent ER/PM junctions are outlined in red. (B) ROIs were drawn around regions of the plasma membrane touching an adjacent cell or facing open space and the mean fluorescence intensity of the indicated proteins within those regions was measured. An enrichment ratio was calculated for each cell by dividing the mean fluorescent intensity at adjacent regions by the mean non-adjacent fluorescent intensities. Student’s t test; P values of P = 0.0010 for TMEM24 and P = 7.12902E-07 for C2CD2. (C and D) Cells expressing TMEM24-eGFP and TMEM24-mCherry were coplated as illustrated by the diagram. (C) TMEM24 expressed in adjacent cells forms symmetrical ER/PM junctions. (D) Gaps in the TMEM24 signal (indicated by arrowheads) in one cell are mirrored by gaps in the signal of the adjacent cell. (E) Diagram illustrating TMEM24 behavior in adjacent cells expressing TMEM24. (F) TMEM24 tagged with <t>GFP</t> at the endogenous locus in IMR32 cells localizes preferentially to the cell–cell interface. (G) Quantification of the enrichment ratio for mean fluorescence of TMEM24 tagged at the endogenous locus (endoTMEM24) at regions of IMR32 cells that face adjacent cells or empty space in the dish. As endoTMEM24 signal may stem from both adjacent cells, the observed fluorescence at the adjacent region has been halved and compared against non-adjacent regions (P = 0.000000003, n = 24 cells). (H) Differentiated IMR32 cells show that endoTMEM24 fluorescence is preferentially localized at sites of cell–cell contact as in the undifferentiated IMR32 cells. In F and H, the plasma membrane was labeled with CellBrite 650 dye. Diagonal lines indicate regions of the micrographs occupied by cells to clearly differentiate these regions from empty spaces. Scale bars = 5 μm.
    Mouse Monoclonal Anti Gfp, supplied by Roche, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Large TMEM24-positive ER/PM junctions at cell–cell interfaces. (A) Left: Spinning disk confocal images of TMEM24-mCherry, C2CD2-eGFP, and JPH4-eGFP in HEK293 cells. Center: <t>plasma</t> <t>membranes</t> labeled with CellBrite 650 dye. Faint diagonal lines have been added to indicate regions of the micrographs occupied by the cells. Right: ER/PM junctions from the left fields with cell-adjacent ER/PM junctions are outlined in red. (B) ROIs were drawn around regions of the plasma membrane touching an adjacent cell or facing open space and the mean fluorescence intensity of the indicated proteins within those regions was measured. An enrichment ratio was calculated for each cell by dividing the mean fluorescent intensity at adjacent regions by the mean non-adjacent fluorescent intensities. Student’s t test; P values of P = 0.0010 for TMEM24 and P = 7.12902E-07 for C2CD2. (C and D) Cells expressing TMEM24-eGFP and TMEM24-mCherry were coplated as illustrated by the diagram. (C) TMEM24 expressed in adjacent cells forms symmetrical ER/PM junctions. (D) Gaps in the TMEM24 signal (indicated by arrowheads) in one cell are mirrored by gaps in the signal of the adjacent cell. (E) Diagram illustrating TMEM24 behavior in adjacent cells expressing TMEM24. (F) TMEM24 tagged with <t>GFP</t> at the endogenous locus in IMR32 cells localizes preferentially to the cell–cell interface. (G) Quantification of the enrichment ratio for mean fluorescence of TMEM24 tagged at the endogenous locus (endoTMEM24) at regions of IMR32 cells that face adjacent cells or empty space in the dish. As endoTMEM24 signal may stem from both adjacent cells, the observed fluorescence at the adjacent region has been halved and compared against non-adjacent regions (P = 0.000000003, n = 24 cells). (H) Differentiated IMR32 cells show that endoTMEM24 fluorescence is preferentially localized at sites of cell–cell contact as in the undifferentiated IMR32 cells. In F and H, the plasma membrane was labeled with CellBrite 650 dye. Diagonal lines indicate regions of the micrographs occupied by cells to clearly differentiate these regions from empty spaces. Scale bars = 5 μm.
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    Image Search Results


    The knockout of Ccdc146 leads to male infertility. A CCDC146 was related to CCDC38 predicted by the STRING database. B CCDC146 interacted with CCDC38. pCSII-MYC-Ccdc146 was transfected into HEK293T cells with pEGFP-C1-Ccdc38, 48 hours after transfection, cells were collected for immunoprecipitation with anti-MYC, and detected by anti-GFP or anti-MYC antibodies, respectively. C CCDC146 co-localized with CCDC38 and γ-TUBULIN in Hela cells. pCSII-MYC-Ccdc146 and pEGFP-C1-Ccdc38 were co-transfected into Hela cells. 24 h after transfection, cells were fixed and stained with anti-MYC and anti-γ-TUBULIN or anti-GFP antibodies, and the nucleus was stained with DAPI. Scale bar: 5 μm. D The expression of Ccdc146 in different tissues of adult mice. GAPDH was used as a loading control. E The expression of Ccdc146 on different days. TUBULIN was used as a loading control. F The schematic illustration of the knockout strategy for generating Ccdc146−/− mice lacking exon 3–7. Primer-F1, Primer-R1, and Primer-R2 were used as the genotyping primers. G Genotyping of Ccdc146−/− mice. H Western blotting of CCDC146 indicated the depletion efficiency in Ccdc146−/− male mice. I Ccdc146−/− male mice were infertile. The Y axis represents the average number of offspring per litter. Data are presented as means ± SEM. two-tailed Student’s t test; ***P < 0·001. J The number of pups per litter of the different crossing possibilities. Data are presented as means ± SEM. two-tailed Student’s t test; ns: no significance; ***P < 0.001. K The sex ratio of the pups of the different crossing possibilities. Data are presented as means ± SEM. two-tailed Student’s t test; ns: no significance; ***P < 0.001. L The size of the Ccdc146+/+ and Ccdc146−/− mice testes were unaffected. M Testis from Ccdc146+/+ and Ccdc146−/− male mice had no obvious difference in weight (n = 5). Data are presented as means ± SEM. two-tailed Student’s t-test; ns: no significance. N The body weight of Ccdc146+/+ and Ccdc146−/− male mice had no obvious difference (n = 5). Data are presented as means ± SEM. two-tailed Student’s t-test; ns: no significance. O The testis/body weight ratios in Ccdc146+/+ and Ccdc146−/− male mice were consistent (n = 5). Data are presented as means ± SEM. two-tailed Student’s t-test; ns no significance

    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: The knockout of Ccdc146 leads to male infertility. A CCDC146 was related to CCDC38 predicted by the STRING database. B CCDC146 interacted with CCDC38. pCSII-MYC-Ccdc146 was transfected into HEK293T cells with pEGFP-C1-Ccdc38, 48 hours after transfection, cells were collected for immunoprecipitation with anti-MYC, and detected by anti-GFP or anti-MYC antibodies, respectively. C CCDC146 co-localized with CCDC38 and γ-TUBULIN in Hela cells. pCSII-MYC-Ccdc146 and pEGFP-C1-Ccdc38 were co-transfected into Hela cells. 24 h after transfection, cells were fixed and stained with anti-MYC and anti-γ-TUBULIN or anti-GFP antibodies, and the nucleus was stained with DAPI. Scale bar: 5 μm. D The expression of Ccdc146 in different tissues of adult mice. GAPDH was used as a loading control. E The expression of Ccdc146 on different days. TUBULIN was used as a loading control. F The schematic illustration of the knockout strategy for generating Ccdc146−/− mice lacking exon 3–7. Primer-F1, Primer-R1, and Primer-R2 were used as the genotyping primers. G Genotyping of Ccdc146−/− mice. H Western blotting of CCDC146 indicated the depletion efficiency in Ccdc146−/− male mice. I Ccdc146−/− male mice were infertile. The Y axis represents the average number of offspring per litter. Data are presented as means ± SEM. two-tailed Student’s t test; ***P < 0·001. J The number of pups per litter of the different crossing possibilities. Data are presented as means ± SEM. two-tailed Student’s t test; ns: no significance; ***P < 0.001. K The sex ratio of the pups of the different crossing possibilities. Data are presented as means ± SEM. two-tailed Student’s t test; ns: no significance; ***P < 0.001. L The size of the Ccdc146+/+ and Ccdc146−/− mice testes were unaffected. M Testis from Ccdc146+/+ and Ccdc146−/− male mice had no obvious difference in weight (n = 5). Data are presented as means ± SEM. two-tailed Student’s t-test; ns: no significance. N The body weight of Ccdc146+/+ and Ccdc146−/− male mice had no obvious difference (n = 5). Data are presented as means ± SEM. two-tailed Student’s t-test; ns: no significance. O The testis/body weight ratios in Ccdc146+/+ and Ccdc146−/− male mice were consistent (n = 5). Data are presented as means ± SEM. two-tailed Student’s t-test; ns no significance

    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: Knock-Out, Transfection, Immunoprecipitation, Staining, Expressing, Western Blot, Two Tailed Test

    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

    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

    CCDC146 interacts with CCDC38 and CCDC42. A CCDC146 interacted with CCDC42. pCSII-MYC-Ccdc146 were transfected into HEK293T cells with pEGFP-C1-Ccdc42 48 hours after transfection; cells were collected for immunoprecipitation with anti-MYC, and detected by anti-GFP or anti-MYC antibodies, respectively. B CCDC146 interacted with CCDC38 and CCDC42 in testis. Co-IP of CCDC38 and CCDC42 with FLAG-CCDC146 from testis lysate using anti-FLAG magnetic beads, followed by western blotting with anti-CCDC38, anti-CCDC42, anti-CCDC146 and anti-FLAG (CCDC146) antibodies. C, D Western blots showing CCDC38, CCDC42, ODF2, IFT88, IFT20, and ODF1 protein levels in lysates from Ccdc146+/+ and Ccdc146−/− mice testes. GAPDH served as a loading control. ODF2, IFT88, and IFT20 protein levels were reduced in Ccdc146−/− testes compared with Ccdc146+/+ testes. Data are presented as means ± SEM. two-tailed Student’s t test; ns: no significance; *P < 0·1; **P < 0·01. E Binding mode of CCDC38 on the CCDC146 predicted by docking. Detailed interaction network between CCDC38 and CCDC146. Key residues of CCDC146 (blue) and CCDC38 (light red) are displayed as sticks. H-bonds are displayed in dash lines. F Binding mode of CCDC42 on the CCDC146 predicted by docking. Detailed interaction network between CCDC42 and CCDC146. Key residues of CCDC146 (blue) and CCDC38 (light yellow) are displayed as sticks. H-bonds are displayed in yellow dashed lines. G Alignment of the mode of CCDC38 and CCDC42 on the CCDC146 predicted by docking. H–I Interactions between CCDC38 and WT or D541, or R547 mutants of truncated CCDC146 were detected by Co-IP assays. J–K Co-IP assays detected interactions between CCDC42 and WT or L232, or Y245 mutants of truncated CCDC146

    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 interacts with CCDC38 and CCDC42. A CCDC146 interacted with CCDC42. pCSII-MYC-Ccdc146 were transfected into HEK293T cells with pEGFP-C1-Ccdc42 48 hours after transfection; cells were collected for immunoprecipitation with anti-MYC, and detected by anti-GFP or anti-MYC antibodies, respectively. B CCDC146 interacted with CCDC38 and CCDC42 in testis. Co-IP of CCDC38 and CCDC42 with FLAG-CCDC146 from testis lysate using anti-FLAG magnetic beads, followed by western blotting with anti-CCDC38, anti-CCDC42, anti-CCDC146 and anti-FLAG (CCDC146) antibodies. C, D Western blots showing CCDC38, CCDC42, ODF2, IFT88, IFT20, and ODF1 protein levels in lysates from Ccdc146+/+ and Ccdc146−/− mice testes. GAPDH served as a loading control. ODF2, IFT88, and IFT20 protein levels were reduced in Ccdc146−/− testes compared with Ccdc146+/+ testes. Data are presented as means ± SEM. two-tailed Student’s t test; ns: no significance; *P < 0·1; **P < 0·01. E Binding mode of CCDC38 on the CCDC146 predicted by docking. Detailed interaction network between CCDC38 and CCDC146. Key residues of CCDC146 (blue) and CCDC38 (light red) are displayed as sticks. H-bonds are displayed in dash lines. F Binding mode of CCDC42 on the CCDC146 predicted by docking. Detailed interaction network between CCDC42 and CCDC146. Key residues of CCDC146 (blue) and CCDC38 (light yellow) are displayed as sticks. H-bonds are displayed in yellow dashed lines. G Alignment of the mode of CCDC38 and CCDC42 on the CCDC146 predicted by docking. H–I Interactions between CCDC38 and WT or D541, or R547 mutants of truncated CCDC146 were detected by Co-IP assays. J–K Co-IP assays detected interactions between CCDC42 and WT or L232, or Y245 mutants of truncated CCDC146

    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, Immunoprecipitation, Co-Immunoprecipitation Assay, Magnetic Beads, Western Blot, Two Tailed Test, Binding Assay

    Large TMEM24-positive ER/PM junctions at cell–cell interfaces. (A) Left: Spinning disk confocal images of TMEM24-mCherry, C2CD2-eGFP, and JPH4-eGFP in HEK293 cells. Center: plasma membranes labeled with CellBrite 650 dye. Faint diagonal lines have been added to indicate regions of the micrographs occupied by the cells. Right: ER/PM junctions from the left fields with cell-adjacent ER/PM junctions are outlined in red. (B) ROIs were drawn around regions of the plasma membrane touching an adjacent cell or facing open space and the mean fluorescence intensity of the indicated proteins within those regions was measured. An enrichment ratio was calculated for each cell by dividing the mean fluorescent intensity at adjacent regions by the mean non-adjacent fluorescent intensities. Student’s t test; P values of P = 0.0010 for TMEM24 and P = 7.12902E-07 for C2CD2. (C and D) Cells expressing TMEM24-eGFP and TMEM24-mCherry were coplated as illustrated by the diagram. (C) TMEM24 expressed in adjacent cells forms symmetrical ER/PM junctions. (D) Gaps in the TMEM24 signal (indicated by arrowheads) in one cell are mirrored by gaps in the signal of the adjacent cell. (E) Diagram illustrating TMEM24 behavior in adjacent cells expressing TMEM24. (F) TMEM24 tagged with GFP at the endogenous locus in IMR32 cells localizes preferentially to the cell–cell interface. (G) Quantification of the enrichment ratio for mean fluorescence of TMEM24 tagged at the endogenous locus (endoTMEM24) at regions of IMR32 cells that face adjacent cells or empty space in the dish. As endoTMEM24 signal may stem from both adjacent cells, the observed fluorescence at the adjacent region has been halved and compared against non-adjacent regions (P = 0.000000003, n = 24 cells). (H) Differentiated IMR32 cells show that endoTMEM24 fluorescence is preferentially localized at sites of cell–cell contact as in the undifferentiated IMR32 cells. In F and H, the plasma membrane was labeled with CellBrite 650 dye. Diagonal lines indicate regions of the micrographs occupied by cells to clearly differentiate these regions from empty spaces. Scale bars = 5 μm.

    Journal: The Journal of Cell Biology

    Article Title: A complex of the lipid transport ER proteins TMEM24 and C2CD2 with band 4.1 at cell–cell contacts

    doi: 10.1083/jcb.202311137

    Figure Lengend Snippet: Large TMEM24-positive ER/PM junctions at cell–cell interfaces. (A) Left: Spinning disk confocal images of TMEM24-mCherry, C2CD2-eGFP, and JPH4-eGFP in HEK293 cells. Center: plasma membranes labeled with CellBrite 650 dye. Faint diagonal lines have been added to indicate regions of the micrographs occupied by the cells. Right: ER/PM junctions from the left fields with cell-adjacent ER/PM junctions are outlined in red. (B) ROIs were drawn around regions of the plasma membrane touching an adjacent cell or facing open space and the mean fluorescence intensity of the indicated proteins within those regions was measured. An enrichment ratio was calculated for each cell by dividing the mean fluorescent intensity at adjacent regions by the mean non-adjacent fluorescent intensities. Student’s t test; P values of P = 0.0010 for TMEM24 and P = 7.12902E-07 for C2CD2. (C and D) Cells expressing TMEM24-eGFP and TMEM24-mCherry were coplated as illustrated by the diagram. (C) TMEM24 expressed in adjacent cells forms symmetrical ER/PM junctions. (D) Gaps in the TMEM24 signal (indicated by arrowheads) in one cell are mirrored by gaps in the signal of the adjacent cell. (E) Diagram illustrating TMEM24 behavior in adjacent cells expressing TMEM24. (F) TMEM24 tagged with GFP at the endogenous locus in IMR32 cells localizes preferentially to the cell–cell interface. (G) Quantification of the enrichment ratio for mean fluorescence of TMEM24 tagged at the endogenous locus (endoTMEM24) at regions of IMR32 cells that face adjacent cells or empty space in the dish. As endoTMEM24 signal may stem from both adjacent cells, the observed fluorescence at the adjacent region has been halved and compared against non-adjacent regions (P = 0.000000003, n = 24 cells). (H) Differentiated IMR32 cells show that endoTMEM24 fluorescence is preferentially localized at sites of cell–cell contact as in the undifferentiated IMR32 cells. In F and H, the plasma membrane was labeled with CellBrite 650 dye. Diagonal lines indicate regions of the micrographs occupied by cells to clearly differentiate these regions from empty spaces. Scale bars = 5 μm.

    Article Snippet: Membranes were probed with mouse anti-GFP (G6539; Sigma-Aldrich) then labeled with LiCor IRDye 680LT Goat α-Mouse (926-68020) and LiCor IRDye 680RD Streptavidin (926-68079).

    Techniques: Labeling, Membrane, Fluorescence, Expressing

    Streptavidin affinity-purification and localization of the identified band 4.1 proteins. Anti-GFP western blots and streptavidin overlay of material affinity-purified on streptavidin bead. Numbers in parenthesis indicate amino acid boundaries of TMEM24 fragments used. Note the high degree of self-biotinylation for each construct. Source data are available for this figure: .

    Journal: The Journal of Cell Biology

    Article Title: A complex of the lipid transport ER proteins TMEM24 and C2CD2 with band 4.1 at cell–cell contacts

    doi: 10.1083/jcb.202311137

    Figure Lengend Snippet: Streptavidin affinity-purification and localization of the identified band 4.1 proteins. Anti-GFP western blots and streptavidin overlay of material affinity-purified on streptavidin bead. Numbers in parenthesis indicate amino acid boundaries of TMEM24 fragments used. Note the high degree of self-biotinylation for each construct. Source data are available for this figure: .

    Article Snippet: Membranes were probed with mouse anti-GFP (G6539; Sigma-Aldrich) then labeled with LiCor IRDye 680LT Goat α-Mouse (926-68020) and LiCor IRDye 680RD Streptavidin (926-68079).

    Techniques: Affinity Purification, Western Blot, Construct