polyclonal anti exoc1 rabbit  (Proteintech)

Journal: eLife

Article Title: EXOC1 plays an integral role in spermatogonia pseudopod elongation and spermatocyte stable syncytium formation in mice

doi: 10.7554/eLife.59759

Figure Lengend Snippet: ( A ) The expression data of each Exocyst subunit were extracted from the open data source, GSE112393 . GC1 is spermatogonia, GC2 and GC3 are preleptotene stages, GC4–GC8 are meiotic spermatocytes, GC9–GC11 are post-meiotic haploid round spermatids, and GC12 is elongating spermatids. All exocyst subunits are expressed in all differentiation stages. Cdh1 , Sall4 , and Neruog3 are known markers of spermatogonia and were used as positive controls. ( B ) Sanger sequencing of PA-Tag and LG3 Linker in Exoc1 PA-N and Exoc1 PA-C alleles. Intended Knock-in sequence was found in each allele. ( C ) Immunofluorescence of Exoc1 +/PA-C adult testis using anti-PA-tag antibody. PA-tagged EXOC1 was observed in the adult male germ cells of interest in this study: undifferentiated spermatogonia (GFRa1 + , Rarγ + ), differentiating spermatogonia (Kit + ), and spermatocyte (γH2AX + ). Scale bars: 100 μm.

Article Snippet: Antibody , Polyclonal anti-Exoc1 (Rabbit) , Proteintech , Cat#11690–1-AP , IF (1:50).

Techniques: Expressing, Sequencing, Knock-In, Immunofluorescence

Journal: eLife

Article Title: EXOC1 plays an integral role in spermatogonia pseudopod elongation and spermatocyte stable syncytium formation in mice

doi: 10.7554/eLife.59759

Figure Lengend Snippet: ( A ) Generation of PA-Tag knock-in mice. In the Exoc1 PA-C allele, the LG3-linker connected PA-tag gene fragment was knocked-in just before the stop codon of Exoc1 using CRISPR-Cas9. In the Exoc1 PA-N allele, the LG3-connected PA-tag gene fragment was knocked in just after the start codon of Exoc1 using CRISPR-Cas12a. Bold letters represent the CRISPR-Cas9 and Cas12a target sequence. ( B ) Western blotting of PA-Tag antibody demonstrated that each C- and N-terminal PA-tagged EXOC1 protein was expressed in the adult testes (n = 2 in each genotype). ( C ) Immunofluorescence with PA-Tag antibody. EXOC1 is observed in every cell in the adult testes. The arrowheads indicate Sertoli cells in which the nucleus is eurochromatin with a large nucleolus. Scale bars: 50 μm. Figure 1—source data 1. Raw data of the in vivo western blot.

Article Snippet: Antibody , Polyclonal anti-Exoc1 (Rabbit) , Proteintech , Cat#11690–1-AP , IF (1:50).

Techniques: Knock-In, CRISPR, Sequencing, Western Blot, Immunofluorescence, In Vivo

Journal: eLife

Article Title: EXOC1 plays an integral role in spermatogonia pseudopod elongation and spermatocyte stable syncytium formation in mice

doi: 10.7554/eLife.59759

Figure Lengend Snippet: The Exoc1 flox strain was from the Exoc1 tm1a(EUCOMM)Hmgu mouse. The Exoc1 tm1a(EUCOMM)Hmgu allele was changed to the Exoc1 tm1c(EUCOMM)Hmgu (is equal to Exoc1 flox ) allele by mating with the Flpe expression mouse (B6;SJL-Tg(ACTFLPe)9205Dym/J) . Exon 4 of Exoc1 was floxed in this allele. Arrows indicate primers for detecting the flox allele.

Article Snippet: Antibody , Polyclonal anti-Exoc1 (Rabbit) , Proteintech , Cat#11690–1-AP , IF (1:50).

Techniques: Expressing

Journal: eLife

Article Title: EXOC1 plays an integral role in spermatogonia pseudopod elongation and spermatocyte stable syncytium formation in mice

doi: 10.7554/eLife.59759

Figure Lengend Snippet: ( A ) PNA-lectin staining of the Exoc1 adult cKO testis. Signals of PNA-lectin, an acrosomal marker was not observed in the Exoc1 cKO testis. Scale bars: 50 μm. ( B ) The macroscopic images for H and E staining. Normal spermatogenesis was not observed in almost all of the seminiferous tubules in the Exoc1 cKO adult testis. Scale bars: 300 μm. ( C ) The mesoscopic images for H and E staining of the Exoc1 cKO testis. Large and circular cells, appearing to be aggregates of syncytia (AGS), containing multiple nuclei were observed in the lumen of seminiferous tubules (arrowheads). Scale bars: 100 μm. Control: Exoc1 flox/wt :: Nanos3 +/Cre adult mice. ( D ) SEM observation of the Exoc1 cKO adult testis. Intercellular bridges (ICBs) (arrows) were found in the syncytia in the control ( Exoc1 flox/wt :: Nanos3 +/Cre ) testis. There were no ICB observed in the AGS of Exoc1 cKO. Scale bars: 5 μm. ( E ) The serial section overlay image of Exoc1 cKO adult testis. There were γ-H2AX (marker of spermatocyte) signals in the AGS nucleus. Scale bars: 50 μm. ( F ) A representative immunofluorescence image of an Exoc1 cKO seminiferous tubule. Kit + syncytia, which are observed in differentiating spermatogonia, have ICB. Scale bars: 50 μm. ( G ) H and E staining and immunofluorescence with CLDN11. CLDN11-positive Sertoli cell tight junction (SCTJ) divides the space between the basal and the luminal compartment, and AGS are present within the luminal compartment (arrowheads). Scale bars: 50 μm.

Article Snippet: Antibody , Polyclonal anti-Exoc1 (Rabbit) , Proteintech , Cat#11690–1-AP , IF (1:50).

Techniques: Staining, Marker, Immunofluorescence

Journal: eLife

Article Title: EXOC1 plays an integral role in spermatogonia pseudopod elongation and spermatocyte stable syncytium formation in mice

doi: 10.7554/eLife.59759

Figure Lengend Snippet: ( A ) Classification reference image of cross-sections of seminiferous tubules to evaluate the incidence of AGS. Cross-sections with AGS indicated by arrowheads were classified as ‘Section containing AGS’. Cross-sections with nuclear condensed sperm (including spermatid and spermatozoa) indicated by arrow were classified as ‘Section containing No AGS But Sperm’. Cross-sections with no AGS or Sperm and empty space in the lumen were classified as ‘Section containing neither AGS nor Sperm’. ( B ) Cross-sections of the seminiferous tubules of Exoc1 cKO and control ( Exoc1 flox/wt :: Nanos3 +/Cre ) adult mice (n = 3 in each genotype) were classified into three categories based on the criteria in ( A ). ( C ) Graphical representation of the results in ( B ). ( D ) Genotypic analysis of blastocysts obtained by in vitro fertilization of sperm from Exoc1 cKO mice with wild-type oocytes revealed no cKO alleles that underwent Cre-LoxP recombination (0/16, n = 2). Control: Exoc1 flox/cKO . M5: Marker 5 (Nippon Genetics).

Article Snippet: Antibody , Polyclonal anti-Exoc1 (Rabbit) , Proteintech , Cat#11690–1-AP , IF (1:50).

Techniques: In Vitro, Marker

Journal: eLife

Article Title: EXOC1 plays an integral role in spermatogonia pseudopod elongation and spermatocyte stable syncytium formation in mice

doi: 10.7554/eLife.59759

Figure Lengend Snippet: Meiotic chromosome synapsis was confirmed by immunofluorescence. In pachytene stage, both the homologous pairing (marked by Sycp3) and synaptonemal complex (marked by Sycp1) were normal in Exoc1 cKO and control (wild-type) autosomal chromosomes (n = 2 in each genotype, 8–17 cells in each mouse).

Article Snippet: Antibody , Polyclonal anti-Exoc1 (Rabbit) , Proteintech , Cat#11690–1-AP , IF (1:50).

Techniques: Immunofluorescence

Journal: eLife

Article Title: EXOC1 plays an integral role in spermatogonia pseudopod elongation and spermatocyte stable syncytium formation in mice

doi: 10.7554/eLife.59759

Figure Lengend Snippet: ( A ) Co-immunoprecipitation of EXOC1-STX2-SNAP23 complex in vitro. FLAG-tagged mouse EXOC1, HA-tagged mouse STX2, and Myc-tagged mouse SNAP23 were co-overexpressed in HEK293T cells. The binding of the three factors was confirmed in all combinations of Co-IP experiments. FLAG-EGFP, V5-mCherry-HA, and E2 crimson-MYC-His were used as negative controls. ( B ) Interaction of EXOC1 with SNAP23 in vivo. PA-tagged EXOC1 was co-immunoprecipitated with endogenous SNAP23 in the adult Exoc1 PA-N testis. The upper and middle panels show short and long period exposure images, respectively. Arrowhead indicates PA-EXOC1. ( C ) The macroscopic images for H and E staining. Sperms were found in frequent seminiferous tubules in adult Snap23 cKO mice. Scale bars: 300 μm. Control: Snap23 flox/wt :: Nanos3 +/Cre mice. ( D ) The mesoscopic images for H and E staining of Snap23 cKO adult testis. Large and circular cells containing multiple nuclei (arrowheads), appearing to be aggregates of syncytia (AGS) were observed. In contrast with Exoc1 cKO, every seminiferous tubule had sperms with elongated nuclei (arrows). Scale bars: 50 μm. Control: Snap23 flox/wt :: Nanos3 +/Cre mice. ( E ) The serial section overlay image of Snap23 cKO testis. Immunofluorescence signals of γ-H2AX were found in AGS. Scale bars: 50 μm. ( F ) PNA-lectin staining of Snap23 cKO testis. PNA-lectin that was used to detect the acrosome is observed in the lumen of the seminiferous tubule of Snap23 cKO testis. Scale bars: 50 μm. Figure 3—source data 1. Raw data of the in vitro immunoprecipitation. Figure 3—source data 2. Raw data of the in vivo immunoprecipitation. Figure 3—source data 3. Occurrence rate of AGS per area in the extracted Section containing AGS.

Article Snippet: Antibody , Polyclonal anti-Exoc1 (Rabbit) , Proteintech , Cat#11690–1-AP , IF (1:50).

Techniques: Immunoprecipitation, In Vitro, Binding Assay, Co-Immunoprecipitation Assay, In Vivo, Staining, Immunofluorescence

Journal: eLife

Article Title: EXOC1 plays an integral role in spermatogonia pseudopod elongation and spermatocyte stable syncytium formation in mice

doi: 10.7554/eLife.59759

Figure Lengend Snippet: ( A ) A representative image of GFRα1 + undifferentiated spermatogonia in Exoc1 cKO and Stx2 KO. Pseudopod elongation was impaired in Exoc1 cKO, but not in Stx2 KO. Scale bars: 10 μm. ( B ) Pseudopod length quantification using sections. Average length of GFRα1 + spermatogonia pseudopods in Exoc1 cKO was shorter than that of Stx2 KO and wild type (n = 3 in each genotype, 25–36 cells in each mouse). *p=0.052, **p=1.8 × 10 −6 , ***p=9.5 × 10 −9 . one-way ANOVA. ( C ) Pseudopod length quantification through whole-mount immunofluorescence staining of adult testes. GFRα1 + spermatogonia with elongated pseudopod (white arrows) were frequently observed in control ( Exoc1 flox/cKO ) mice, whereas they were rarely observed in Exoc1 cKO mice. Scale bars: 30 μm. ( D ) Measurement of the length of pseudopod of A single GFRα1 + cells based on whole-mount immunofluorescence images (n = 3 in each genotype, 20 cells in each mouse). *p=7.2 × 10 −17 , Student’s t-test. Control: Exoc1 flox/cKO . ( E, F ) Measurement of intercellular length in connected A pair (n = 3 in each genotype, 20 intercellular distances in each mouse) or A aligned (n = 3 in each genotype, 14–19 intercellular distances in each mouse) based on whole-mount immunofluorescence images. *p=3.6 × 10 −12 , **p=0.00014, Student’s t-test. Control: Exoc1 flox/cKO . ( G ) A representative image of active-Rac1 in GFRα1 + spermatogonia of Exoc1 cKO adult testis. In control mice ( Exoc1 flox/wt :: Nanos3 +/Cre ), active-Rac1 signal was lower than the detection limit. Non-polar active-Rac1 signal was detected in Exoc1 cKO adult testis. Scale bars: 5 μm. ( H ) Quantification of signal intensity of active-Rac1 in GFRα1 + spermatogonia based on immunostaining images. The average intensity in each cell is higher in the Exoc1 cKO group than that in the control group (n = 3 in genotype, 8–10 cells in each mouse). *p=0.000036, Student’s t-test. Control: Exoc1 flox/flox . Figure 4—source data 1. Measurement of the length of the pseudopodia of GFRα1+ cells in section observation. Figure 4—source data 2. Measurement of the length of the pseudopodia of GFRα1+ cells in whole-mount observation. Figure 4—source data 3. Intensity of active Rac1 signal in each cell.

Article Snippet: Antibody , Polyclonal anti-Exoc1 (Rabbit) , Proteintech , Cat#11690–1-AP , IF (1:50).

Techniques: Immunofluorescence, Staining, Immunostaining

Journal: eLife

Article Title: EXOC1 plays an integral role in spermatogonia pseudopod elongation and spermatocyte stable syncytium formation in mice

doi: 10.7554/eLife.59759

Figure Lengend Snippet: ( A ) Representative immunofluorescence image of adult Exoc1 cKO seminiferous tubule. GFRα1 + spermatogonia (arrow) density in the cKO was higher than that in control mice. RARγ + spermatogonia (arrowhead) density decreased in the cKO. Control: Exoc1 flox/flox mice. ( B ) The number of spermatogonia in a cross-section of seminiferous tubule (n = 3 in each genotype, 46–87 sections in each mouse). The number of GFRα1 + cell per section was significantly increased in the cKO. The number of RARγ + cell in the cKO was significantly smaller than that in control ( Exoc1 flox/flox ) mice. *p=1.9 × 10 −7 , **p=3.6 × 10 −9 , ***p=0.035, Student’s t-test. Figure 5—source data 1. The number of GFRα1+ and RARγ+ cells. Figure 5—source data 2. The number of c-Kit+ cells.

Article Snippet: Antibody , Polyclonal anti-Exoc1 (Rabbit) , Proteintech , Cat#11690–1-AP , IF (1:50).

Techniques: Immunofluorescence

Journal: eLife

Article Title: EXOC1 plays an integral role in spermatogonia pseudopod elongation and spermatocyte stable syncytium formation in mice

doi: 10.7554/eLife.59759

Figure Lengend Snippet: The number of Kit + spermatogonia in each one cross-section of seminiferous tubule in adult mice (n = 3 in each genotype, 20 sections in each mouse). *p=0.000012, Student’s t-test. Control: Exoc1 flox/flox .

Article Snippet: Antibody , Polyclonal anti-Exoc1 (Rabbit) , Proteintech , Cat#11690–1-AP , IF (1:50).

Techniques:

Journal: eLife

Article Title: EXOC1 plays an integral role in spermatogonia pseudopod elongation and spermatocyte stable syncytium formation in mice

doi: 10.7554/eLife.59759

Figure Lengend Snippet:

Article Snippet: Antibody , Polyclonal anti-Exoc1 (Rabbit) , Proteintech , Cat#11690–1-AP , IF (1:50).

Techniques: Recombinant, Expressing, Plasmid Preparation, Sequencing, Software, Staining

Journal: eLife

Article Title: EXOC1 plays an integral role in spermatogonia pseudopod elongation and spermatocyte stable syncytium formation in mice

doi: 10.7554/eLife.59759

Figure Lengend Snippet: ( A ) The expression data of each Exocyst subunit were extracted from the open data source, GSE112393 . GC1 is spermatogonia, GC2 and GC3 are preleptotene stages, GC4–GC8 are meiotic spermatocytes, GC9–GC11 are post-meiotic haploid round spermatids, and GC12 is elongating spermatids. All exocyst subunits are expressed in all differentiation stages. Cdh1 , Sall4 , and Neruog3 are known markers of spermatogonia and were used as positive controls. ( B ) Sanger sequencing of PA-Tag and LG3 Linker in Exoc1 PA-N and Exoc1 PA-C alleles. Intended Knock-in sequence was found in each allele. ( C ) Immunofluorescence of Exoc1 +/PA-C adult testis using anti-PA-tag antibody. PA-tagged EXOC1 was observed in the adult male germ cells of interest in this study: undifferentiated spermatogonia (GFRa1 + , Rarγ + ), differentiating spermatogonia (Kit + ), and spermatocyte (γH2AX + ). Scale bars: 100 μm.

Article Snippet: Antibody , Polyclonal anti-Exoc1 (Rabbit) , Atlas Antibodies , Cat#HPA037706 , IF (1:50).

Techniques: Expressing, Sequencing, Knock-In, Immunofluorescence

Journal: eLife

Article Title: EXOC1 plays an integral role in spermatogonia pseudopod elongation and spermatocyte stable syncytium formation in mice

doi: 10.7554/eLife.59759

Figure Lengend Snippet: ( A ) Generation of PA-Tag knock-in mice. In the Exoc1 PA-C allele, the LG3-linker connected PA-tag gene fragment was knocked-in just before the stop codon of Exoc1 using CRISPR-Cas9. In the Exoc1 PA-N allele, the LG3-connected PA-tag gene fragment was knocked in just after the start codon of Exoc1 using CRISPR-Cas12a. Bold letters represent the CRISPR-Cas9 and Cas12a target sequence. ( B ) Western blotting of PA-Tag antibody demonstrated that each C- and N-terminal PA-tagged EXOC1 protein was expressed in the adult testes (n = 2 in each genotype). ( C ) Immunofluorescence with PA-Tag antibody. EXOC1 is observed in every cell in the adult testes. The arrowheads indicate Sertoli cells in which the nucleus is eurochromatin with a large nucleolus. Scale bars: 50 μm. Figure 1—source data 1. Raw data of the in vivo western blot.

Article Snippet: Antibody , Polyclonal anti-Exoc1 (Rabbit) , Atlas Antibodies , Cat#HPA037706 , IF (1:50).

Techniques: Knock-In, CRISPR, Sequencing, Western Blot, Immunofluorescence, In Vivo

Journal: eLife

Article Title: EXOC1 plays an integral role in spermatogonia pseudopod elongation and spermatocyte stable syncytium formation in mice

doi: 10.7554/eLife.59759

Figure Lengend Snippet: The Exoc1 flox strain was from the Exoc1 tm1a(EUCOMM)Hmgu mouse. The Exoc1 tm1a(EUCOMM)Hmgu allele was changed to the Exoc1 tm1c(EUCOMM)Hmgu (is equal to Exoc1 flox ) allele by mating with the Flpe expression mouse (B6;SJL-Tg(ACTFLPe)9205Dym/J) . Exon 4 of Exoc1 was floxed in this allele. Arrows indicate primers for detecting the flox allele.

Article Snippet: Antibody , Polyclonal anti-Exoc1 (Rabbit) , Atlas Antibodies , Cat#HPA037706 , IF (1:50).

Techniques: Expressing

Journal: eLife

Article Title: EXOC1 plays an integral role in spermatogonia pseudopod elongation and spermatocyte stable syncytium formation in mice

doi: 10.7554/eLife.59759

Figure Lengend Snippet: ( A ) PNA-lectin staining of the Exoc1 adult cKO testis. Signals of PNA-lectin, an acrosomal marker was not observed in the Exoc1 cKO testis. Scale bars: 50 μm. ( B ) The macroscopic images for H and E staining. Normal spermatogenesis was not observed in almost all of the seminiferous tubules in the Exoc1 cKO adult testis. Scale bars: 300 μm. ( C ) The mesoscopic images for H and E staining of the Exoc1 cKO testis. Large and circular cells, appearing to be aggregates of syncytia (AGS), containing multiple nuclei were observed in the lumen of seminiferous tubules (arrowheads). Scale bars: 100 μm. Control: Exoc1 flox/wt :: Nanos3 +/Cre adult mice. ( D ) SEM observation of the Exoc1 cKO adult testis. Intercellular bridges (ICBs) (arrows) were found in the syncytia in the control ( Exoc1 flox/wt :: Nanos3 +/Cre ) testis. There were no ICB observed in the AGS of Exoc1 cKO. Scale bars: 5 μm. ( E ) The serial section overlay image of Exoc1 cKO adult testis. There were γ-H2AX (marker of spermatocyte) signals in the AGS nucleus. Scale bars: 50 μm. ( F ) A representative immunofluorescence image of an Exoc1 cKO seminiferous tubule. Kit + syncytia, which are observed in differentiating spermatogonia, have ICB. Scale bars: 50 μm. ( G ) H and E staining and immunofluorescence with CLDN11. CLDN11-positive Sertoli cell tight junction (SCTJ) divides the space between the basal and the luminal compartment, and AGS are present within the luminal compartment (arrowheads). Scale bars: 50 μm.

Article Snippet: Antibody , Polyclonal anti-Exoc1 (Rabbit) , Atlas Antibodies , Cat#HPA037706 , IF (1:50).

Techniques: Staining, Marker, Immunofluorescence

Journal: eLife

Article Title: EXOC1 plays an integral role in spermatogonia pseudopod elongation and spermatocyte stable syncytium formation in mice

doi: 10.7554/eLife.59759

Figure Lengend Snippet: ( A ) Classification reference image of cross-sections of seminiferous tubules to evaluate the incidence of AGS. Cross-sections with AGS indicated by arrowheads were classified as ‘Section containing AGS’. Cross-sections with nuclear condensed sperm (including spermatid and spermatozoa) indicated by arrow were classified as ‘Section containing No AGS But Sperm’. Cross-sections with no AGS or Sperm and empty space in the lumen were classified as ‘Section containing neither AGS nor Sperm’. ( B ) Cross-sections of the seminiferous tubules of Exoc1 cKO and control ( Exoc1 flox/wt :: Nanos3 +/Cre ) adult mice (n = 3 in each genotype) were classified into three categories based on the criteria in ( A ). ( C ) Graphical representation of the results in ( B ). ( D ) Genotypic analysis of blastocysts obtained by in vitro fertilization of sperm from Exoc1 cKO mice with wild-type oocytes revealed no cKO alleles that underwent Cre-LoxP recombination (0/16, n = 2). Control: Exoc1 flox/cKO . M5: Marker 5 (Nippon Genetics).

Article Snippet: Antibody , Polyclonal anti-Exoc1 (Rabbit) , Atlas Antibodies , Cat#HPA037706 , IF (1:50).

Techniques: In Vitro, Marker

Journal: eLife

Article Title: EXOC1 plays an integral role in spermatogonia pseudopod elongation and spermatocyte stable syncytium formation in mice

doi: 10.7554/eLife.59759

Figure Lengend Snippet: Meiotic chromosome synapsis was confirmed by immunofluorescence. In pachytene stage, both the homologous pairing (marked by Sycp3) and synaptonemal complex (marked by Sycp1) were normal in Exoc1 cKO and control (wild-type) autosomal chromosomes (n = 2 in each genotype, 8–17 cells in each mouse).

Article Snippet: Antibody , Polyclonal anti-Exoc1 (Rabbit) , Atlas Antibodies , Cat#HPA037706 , IF (1:50).

Techniques: Immunofluorescence

Journal: eLife

Article Title: EXOC1 plays an integral role in spermatogonia pseudopod elongation and spermatocyte stable syncytium formation in mice

doi: 10.7554/eLife.59759

Figure Lengend Snippet: ( A ) Co-immunoprecipitation of EXOC1-STX2-SNAP23 complex in vitro. FLAG-tagged mouse EXOC1, HA-tagged mouse STX2, and Myc-tagged mouse SNAP23 were co-overexpressed in HEK293T cells. The binding of the three factors was confirmed in all combinations of Co-IP experiments. FLAG-EGFP, V5-mCherry-HA, and E2 crimson-MYC-His were used as negative controls. ( B ) Interaction of EXOC1 with SNAP23 in vivo. PA-tagged EXOC1 was co-immunoprecipitated with endogenous SNAP23 in the adult Exoc1 PA-N testis. The upper and middle panels show short and long period exposure images, respectively. Arrowhead indicates PA-EXOC1. ( C ) The macroscopic images for H and E staining. Sperms were found in frequent seminiferous tubules in adult Snap23 cKO mice. Scale bars: 300 μm. Control: Snap23 flox/wt :: Nanos3 +/Cre mice. ( D ) The mesoscopic images for H and E staining of Snap23 cKO adult testis. Large and circular cells containing multiple nuclei (arrowheads), appearing to be aggregates of syncytia (AGS) were observed. In contrast with Exoc1 cKO, every seminiferous tubule had sperms with elongated nuclei (arrows). Scale bars: 50 μm. Control: Snap23 flox/wt :: Nanos3 +/Cre mice. ( E ) The serial section overlay image of Snap23 cKO testis. Immunofluorescence signals of γ-H2AX were found in AGS. Scale bars: 50 μm. ( F ) PNA-lectin staining of Snap23 cKO testis. PNA-lectin that was used to detect the acrosome is observed in the lumen of the seminiferous tubule of Snap23 cKO testis. Scale bars: 50 μm. Figure 3—source data 1. Raw data of the in vitro immunoprecipitation. Figure 3—source data 2. Raw data of the in vivo immunoprecipitation. Figure 3—source data 3. Occurrence rate of AGS per area in the extracted Section containing AGS.

Article Snippet: Antibody , Polyclonal anti-Exoc1 (Rabbit) , Atlas Antibodies , Cat#HPA037706 , IF (1:50).

Techniques: Immunoprecipitation, In Vitro, Binding Assay, Co-Immunoprecipitation Assay, In Vivo, Staining, Immunofluorescence

Journal: eLife

Article Title: EXOC1 plays an integral role in spermatogonia pseudopod elongation and spermatocyte stable syncytium formation in mice

doi: 10.7554/eLife.59759

Figure Lengend Snippet: ( A ) A representative image of GFRα1 + undifferentiated spermatogonia in Exoc1 cKO and Stx2 KO. Pseudopod elongation was impaired in Exoc1 cKO, but not in Stx2 KO. Scale bars: 10 μm. ( B ) Pseudopod length quantification using sections. Average length of GFRα1 + spermatogonia pseudopods in Exoc1 cKO was shorter than that of Stx2 KO and wild type (n = 3 in each genotype, 25–36 cells in each mouse). *p=0.052, **p=1.8 × 10 −6 , ***p=9.5 × 10 −9 . one-way ANOVA. ( C ) Pseudopod length quantification through whole-mount immunofluorescence staining of adult testes. GFRα1 + spermatogonia with elongated pseudopod (white arrows) were frequently observed in control ( Exoc1 flox/cKO ) mice, whereas they were rarely observed in Exoc1 cKO mice. Scale bars: 30 μm. ( D ) Measurement of the length of pseudopod of A single GFRα1 + cells based on whole-mount immunofluorescence images (n = 3 in each genotype, 20 cells in each mouse). *p=7.2 × 10 −17 , Student’s t-test. Control: Exoc1 flox/cKO . ( E, F ) Measurement of intercellular length in connected A pair (n = 3 in each genotype, 20 intercellular distances in each mouse) or A aligned (n = 3 in each genotype, 14–19 intercellular distances in each mouse) based on whole-mount immunofluorescence images. *p=3.6 × 10 −12 , **p=0.00014, Student’s t-test. Control: Exoc1 flox/cKO . ( G ) A representative image of active-Rac1 in GFRα1 + spermatogonia of Exoc1 cKO adult testis. In control mice ( Exoc1 flox/wt :: Nanos3 +/Cre ), active-Rac1 signal was lower than the detection limit. Non-polar active-Rac1 signal was detected in Exoc1 cKO adult testis. Scale bars: 5 μm. ( H ) Quantification of signal intensity of active-Rac1 in GFRα1 + spermatogonia based on immunostaining images. The average intensity in each cell is higher in the Exoc1 cKO group than that in the control group (n = 3 in genotype, 8–10 cells in each mouse). *p=0.000036, Student’s t-test. Control: Exoc1 flox/flox . Figure 4—source data 1. Measurement of the length of the pseudopodia of GFRα1+ cells in section observation. Figure 4—source data 2. Measurement of the length of the pseudopodia of GFRα1+ cells in whole-mount observation. Figure 4—source data 3. Intensity of active Rac1 signal in each cell.

Article Snippet: Antibody , Polyclonal anti-Exoc1 (Rabbit) , Atlas Antibodies , Cat#HPA037706 , IF (1:50).

Techniques: Immunofluorescence, Staining, Immunostaining

Journal: eLife

Article Title: EXOC1 plays an integral role in spermatogonia pseudopod elongation and spermatocyte stable syncytium formation in mice

doi: 10.7554/eLife.59759

Figure Lengend Snippet: ( A ) Representative immunofluorescence image of adult Exoc1 cKO seminiferous tubule. GFRα1 + spermatogonia (arrow) density in the cKO was higher than that in control mice. RARγ + spermatogonia (arrowhead) density decreased in the cKO. Control: Exoc1 flox/flox mice. ( B ) The number of spermatogonia in a cross-section of seminiferous tubule (n = 3 in each genotype, 46–87 sections in each mouse). The number of GFRα1 + cell per section was significantly increased in the cKO. The number of RARγ + cell in the cKO was significantly smaller than that in control ( Exoc1 flox/flox ) mice. *p=1.9 × 10 −7 , **p=3.6 × 10 −9 , ***p=0.035, Student’s t-test. Figure 5—source data 1. The number of GFRα1+ and RARγ+ cells. Figure 5—source data 2. The number of c-Kit+ cells.

Article Snippet: Antibody , Polyclonal anti-Exoc1 (Rabbit) , Atlas Antibodies , Cat#HPA037706 , IF (1:50).

Techniques: Immunofluorescence

Journal: eLife

Article Title: EXOC1 plays an integral role in spermatogonia pseudopod elongation and spermatocyte stable syncytium formation in mice

doi: 10.7554/eLife.59759

Figure Lengend Snippet: The number of Kit + spermatogonia in each one cross-section of seminiferous tubule in adult mice (n = 3 in each genotype, 20 sections in each mouse). *p=0.000012, Student’s t-test. Control: Exoc1 flox/flox .

Article Snippet: Antibody , Polyclonal anti-Exoc1 (Rabbit) , Atlas Antibodies , Cat#HPA037706 , IF (1:50).

Techniques:

Journal: eLife

Article Title: EXOC1 plays an integral role in spermatogonia pseudopod elongation and spermatocyte stable syncytium formation in mice

doi: 10.7554/eLife.59759

Figure Lengend Snippet:

Article Snippet: Antibody , Polyclonal anti-Exoc1 (Rabbit) , Atlas Antibodies , Cat#HPA037706 , IF (1:50).

Techniques: Recombinant, Expressing, Plasmid Preparation, Sequencing, Software, Staining

Journal: PLoS Pathogens

Article Title: Shigella hijacks the exocyst to cluster macropinosomes for efficient vacuolar escape

doi: 10.1371/journal.ppat.1008822

Figure Lengend Snippet: Time-lapse microscopic analysis of recruitment of the exocyst subunits (grey) (A) Sec3, (B) Sec5 and (C) Exo70 in transfected HeLa cells. Galectin3-mOrange (red) was used as a marker of vacuolar rupture. Images were recorded every minute and z-projections of representative entry sites are shown. The blue arrowheads indicate the IAMs enriched with the respective exocyst subunits. The pink arrowheads in A-B indicate the sites where the respective exocyst subunits localized at close proximity to the BCV. Scale bars are 5 μm. (D) Analysis of Exo70 (green) and Sec5 (red) recruitment by time-lapse microscope indicated that Exo70 was present at the IAMs (indicated by the yellow arrowheads) while Sec5 was localized at the IAMs and BCV (indicated by the white arrowheads and white arrows, respectively). Scale bar is 5 μm.

Article Snippet: For immunofluorescence using Sec3, Sec5, Exo70 antibodies, cells were permeabilized and blocked with 0.25% saponin in the presence of 0.5% bovine serum albumin, 10% fetal bovine serum in PBS buffer for 1 hour at room temperature prior to primary antibody staining using anti-rabbit anti-Sec3 antibody (Proteintech-#11690-1-AP) in 1:100 dilution or anti-mouse anti-Sec5/Exoc2 antibody (Proteintech-#66011-1-Ig) in 1:200 dilution or anti-mouse anti-Exo70 antibody (Sigma-#WH0023265M1) used at 1:200 dilution.

Techniques: Transfection, Marker, Microscopy