Journal: Frontiers in Immunology

Article Title: HSP90AA1 interacts with CSFV NS5A protein and regulates CSFV replication via the JAK/STAT and NF-κB signaling pathway

doi: 10.3389/fimmu.2022.1031868

Figure Lengend Snippet: HSP90AA1 over-expression activates STAT1 and P65 in PK-15 and 3D4/2 cells. (A) HSP90AA1 over-expression affected the phosphorylation of STAT1 and nuclear translocation of p-STAT1 in PK-15 and 3D4/2 cells were observed by laser confocal microscopy. (B) HSP90AA1 over-expression affected the nuclear translocation of p-P65 in PK-15 and 3D4/2 cells were observed by laser confocal microscopy.

Article Snippet: Primary antibodies include rabbit anti-p-STAT1 pAb (Bioss, bs-3427R), rabbit-anti-p-P65 pAb (Affinity Biosciences, AF2006) and mouse anti-Flag mAb.

Techniques: Over Expression, Translocation Assay, Confocal Microscopy

Journal: Frontiers in Immunology

Article Title: HSP90AA1 interacts with CSFV NS5A protein and regulates CSFV replication via the JAK/STAT and NF-κB signaling pathway

doi: 10.3389/fimmu.2022.1031868

Figure Lengend Snippet: CSFV infection antagonizes the activation of HSP90AA1 on JAK/STAT pathway. (A, B) Western blot for JAK1, STAT1, p-JAK1, p-STAT1, OAS2, ISG-15 and HSP90AA1 expression in HSP90AA1-overexpression or knock-downed PK-15 and 3D4/2 cells. Cells were infected with CSFV (MOI=1) after transfection p3×Flag-HSP90AA1 or siHSP90AA1. The cells were not infected with CSFV as a control. Cells were harvested at 24hpi and 48hpi served to Western blot. The relative levels of proteins were estimated by histograms representing density reading of the gel bands with Image J, and the ratios were calculated relative to tubulin control. (*p < 0.05, **p < 0.01, ***p < 0.001 and ****p < 0.0001 calculated using two-way ANOVA, ns, not significant).

Article Snippet: Primary antibodies include rabbit anti-p-STAT1 pAb (Bioss, bs-3427R), rabbit-anti-p-P65 pAb (Affinity Biosciences, AF2006) and mouse anti-Flag mAb.

Techniques: Infection, Activation Assay, Western Blot, Expressing, Over Expression, Transfection

Journal: Biology of Reproduction

Article Title: Cell-autonomous requirement for mammalian target of rapamycin ( Mtor ) in spermatogonial proliferation and differentiation in the mouse †

doi: 10.1093/biolre/iox022

Figure Lengend Snippet: Antibodies

Article Snippet: Images for quantitation were acquired with a Fluoview FV1000 laser scanning confocal microscope (Olympus America). table ft1 table-wrap mode="anchored" t5 Table 1. caption a7 Protein Vendor (Catalog Number) Dilution STRA8 Abcam (ab49602) 1:3000 ZBTB16 Santa Cruz (sc-22839) 1:500 c-PARP1 Cell Signaling Technology (#9544) 1:100 DDX4 R&D Systems (AF2030), Abcam (ab13840) 1:800, 1:1000 GATA4 Santa Cruz (sc-1237) 1:100 GFRA1 Proteintech (AF560) 1:800 KIT Cell Signaling Technology (#3074) 1:1000 MTOR Cell Signaling Technology (#2983P) 1:400 p-RPS6 Cell Signaling Technology (#5364P) 1:800 TRA98 Abcam (ab82427) 1:1000 Open in a separate window Antibodies

Techniques:

Journal: Biology of Reproduction

Article Title: Cell-autonomous requirement for mammalian target of rapamycin ( Mtor ) in spermatogonial proliferation and differentiation in the mouse †

doi: 10.1093/biolre/iox022

Figure Lengend Snippet: Adult (P150) Mtor KO testes contain only undifferentiated spermatogonia. Spermatogonia are labeled for the undifferentiated markers GFRA1 (A, B) and ZBTB16 (C, D), the differentiating marker KIT (E, F), or the proliferating marker MKI67 (A, B). Examples of spermatogonia positive for each marker are indicated by white arrowheads. TRA98 (in red) was used to label all germ cells in (A, B, E–H). Nuclei are labeled (in blue) with DAPI, and the seminiferous tubules are outlined with dashed white lines. Scale bar (in A) = 50 μm.

Article Snippet: Images for quantitation were acquired with a Fluoview FV1000 laser scanning confocal microscope (Olympus America). table ft1 table-wrap mode="anchored" t5 Table 1. caption a7 Protein Vendor (Catalog Number) Dilution STRA8 Abcam (ab49602) 1:3000 ZBTB16 Santa Cruz (sc-22839) 1:500 c-PARP1 Cell Signaling Technology (#9544) 1:100 DDX4 R&D Systems (AF2030), Abcam (ab13840) 1:800, 1:1000 GATA4 Santa Cruz (sc-1237) 1:100 GFRA1 Proteintech (AF560) 1:800 KIT Cell Signaling Technology (#3074) 1:1000 MTOR Cell Signaling Technology (#2983P) 1:400 p-RPS6 Cell Signaling Technology (#5364P) 1:800 TRA98 Abcam (ab82427) 1:1000 Open in a separate window Antibodies

Techniques: Labeling, Marker

Journal: Biology of Reproduction

Article Title: Cell-autonomous requirement for mammalian target of rapamycin ( Mtor ) in spermatogonial proliferation and differentiation in the mouse †

doi: 10.1093/biolre/iox022

Figure Lengend Snippet: Only undifferentiated spermatogonia are retained in P18 Mtor KO testes. In each row, representative images of immunofluorescence for specific spermatogonial fate marker proteins (colors indicate the specific marker on each panel), including ZBTB16 (A, B), GFRA1 (C, D), and KIT (E, F) as well as the proliferation marker MKI67 (G, H) along with TRA98 to mark all germ cells. Nuclei are labeled with DAPI, in blue. The accompanying quantitation for WT and KO testes is presented alongside each panel. Asterisks denote statistical significance (**P < 0.01). Error bars indicate one standard deviation. Scale bar (in A) = 50 μm.

Article Snippet: Images for quantitation were acquired with a Fluoview FV1000 laser scanning confocal microscope (Olympus America). table ft1 table-wrap mode="anchored" t5 Table 1. caption a7 Protein Vendor (Catalog Number) Dilution STRA8 Abcam (ab49602) 1:3000 ZBTB16 Santa Cruz (sc-22839) 1:500 c-PARP1 Cell Signaling Technology (#9544) 1:100 DDX4 R&D Systems (AF2030), Abcam (ab13840) 1:800, 1:1000 GATA4 Santa Cruz (sc-1237) 1:100 GFRA1 Proteintech (AF560) 1:800 KIT Cell Signaling Technology (#3074) 1:1000 MTOR Cell Signaling Technology (#2983P) 1:400 p-RPS6 Cell Signaling Technology (#5364P) 1:800 TRA98 Abcam (ab82427) 1:1000 Open in a separate window Antibodies

Techniques: Immunofluorescence, Marker, Labeling, Quantitation Assay, Standard Deviation

Journal: Biology of Reproduction

Article Title: Cell-autonomous requirement for mammalian target of rapamycin ( Mtor ) in spermatogonial proliferation and differentiation in the mouse †

doi: 10.1093/biolre/iox022

Figure Lengend Snippet: Spermatogonial differentiation is blocked in Mtor KO mice at P8. In each row, representative images of immunofluorescence for specific spermatogonial fate marker proteins (colors indicate the specific marker on each panel), including ZBTB16 (A, B), GFRA1 (C, D), KIT (E, F), and STRA8 (G, H) along with DDX4 to mark all germ cells (in red, A, B and E–H). Nuclei are labeled (in A–F) with DAPI, in blue. The accompanying quantitation for WT and KO testes is presented alongside each panel. Asterisks denote statistical significance (*P < 0.05, **P < 0.01). Error bars indicate one standard deviation. Scale bar (in A) = 50 μm.

Article Snippet: Images for quantitation were acquired with a Fluoview FV1000 laser scanning confocal microscope (Olympus America). table ft1 table-wrap mode="anchored" t5 Table 1. caption a7 Protein Vendor (Catalog Number) Dilution STRA8 Abcam (ab49602) 1:3000 ZBTB16 Santa Cruz (sc-22839) 1:500 c-PARP1 Cell Signaling Technology (#9544) 1:100 DDX4 R&D Systems (AF2030), Abcam (ab13840) 1:800, 1:1000 GATA4 Santa Cruz (sc-1237) 1:100 GFRA1 Proteintech (AF560) 1:800 KIT Cell Signaling Technology (#3074) 1:1000 MTOR Cell Signaling Technology (#2983P) 1:400 p-RPS6 Cell Signaling Technology (#5364P) 1:800 TRA98 Abcam (ab82427) 1:1000 Open in a separate window Antibodies

Techniques: Immunofluorescence, Marker, Labeling, Quantitation Assay, Standard Deviation

Journal: Biology of Reproduction

Article Title: Cell-autonomous requirement for mammalian target of rapamycin ( Mtor ) in spermatogonial proliferation and differentiation in the mouse †

doi: 10.1093/biolre/iox022

Figure Lengend Snippet: Antibodies

Article Snippet: Images for quantitation were acquired with a Fluoview FV1000 laser scanning confocal microscope (Olympus America). table ft1 table-wrap mode="anchored" t5 Table 1. caption a7 Protein Vendor (Catalog Number) Dilution STRA8 Abcam (ab49602) 1:3000 ZBTB16 Santa Cruz (sc-22839) 1:500 c-PARP1 Cell Signaling Technology (#9544) 1:100 DDX4 R&D Systems (AF2030), Abcam (ab13840) 1:800, 1:1000 GATA4 Santa Cruz (sc-1237) 1:100 GFRA1 Proteintech (AF560) 1:800 KIT Cell Signaling Technology (#3074) 1:1000 MTOR Cell Signaling Technology (#2983P) 1:400 p-RPS6 Cell Signaling Technology (#5364P) 1:800 TRA98 Abcam (ab82427) 1:1000 Open in a separate window Antibodies Cauda epididymal sperm counts Cauda epididymides were dissected immediately following euthanasia.

Techniques:

Journal: Biology of Reproduction

Article Title: Cell-autonomous requirement for mammalian target of rapamycin ( Mtor ) in spermatogonial proliferation and differentiation in the mouse †

doi: 10.1093/biolre/iox022

Figure Lengend Snippet: Confirming MTOR deletion in KO testes. (A–F) Immunostaining for MTOR (in green) reveals a clear signal in DDX4+ spermatogonia (in red) from WT but not KO mice. Co-stained cells are shown in yellow. (G–L) Phosphorylated RPS6 (in green) is present in P8 WT DDX4+ spermatogonia (in red), but not in KO spermatogonia. Scale bar (in A) = 50 μm.

Article Snippet: Images for quantitation were acquired with a Fluoview FV1000 laser scanning confocal microscope (Olympus America). table ft1 table-wrap mode="anchored" t5 Table 1. caption a7 Protein Vendor (Catalog Number) Dilution STRA8 Abcam (ab49602) 1:3000 ZBTB16 Santa Cruz (sc-22839) 1:500 c-PARP1 Cell Signaling Technology (#9544) 1:100 DDX4 R&D Systems (AF2030), Abcam (ab13840) 1:800, 1:1000 GATA4 Santa Cruz (sc-1237) 1:100 GFRA1 Proteintech (AF560) 1:800 KIT Cell Signaling Technology (#3074) 1:1000 MTOR Cell Signaling Technology (#2983P) 1:400 p-RPS6 Cell Signaling Technology (#5364P) 1:800 TRA98 Abcam (ab82427) 1:1000 Open in a separate window Antibodies Cauda epididymal sperm counts Cauda epididymides were dissected immediately following euthanasia.

Techniques: Immunostaining, Staining

Journal: Biology of Reproduction

Article Title: Cell-autonomous requirement for mammalian target of rapamycin ( Mtor ) in spermatogonial proliferation and differentiation in the mouse †

doi: 10.1093/biolre/iox022

Figure Lengend Snippet: Adult (P150) Mtor KO testes contain only undifferentiated spermatogonia. Spermatogonia are labeled for the undifferentiated markers GFRA1 (A, B) and ZBTB16 (C, D), the differentiating marker KIT (E, F), or the proliferating marker MKI67 (A, B). Examples of spermatogonia positive for each marker are indicated by white arrowheads. TRA98 (in red) was used to label all germ cells in (A, B, E–H). Nuclei are labeled (in blue) with DAPI, and the seminiferous tubules are outlined with dashed white lines. Scale bar (in A) = 50 μm.

Article Snippet: Images for quantitation were acquired with a Fluoview FV1000 laser scanning confocal microscope (Olympus America). table ft1 table-wrap mode="anchored" t5 Table 1. caption a7 Protein Vendor (Catalog Number) Dilution STRA8 Abcam (ab49602) 1:3000 ZBTB16 Santa Cruz (sc-22839) 1:500 c-PARP1 Cell Signaling Technology (#9544) 1:100 DDX4 R&D Systems (AF2030), Abcam (ab13840) 1:800, 1:1000 GATA4 Santa Cruz (sc-1237) 1:100 GFRA1 Proteintech (AF560) 1:800 KIT Cell Signaling Technology (#3074) 1:1000 MTOR Cell Signaling Technology (#2983P) 1:400 p-RPS6 Cell Signaling Technology (#5364P) 1:800 TRA98 Abcam (ab82427) 1:1000 Open in a separate window Antibodies Cauda epididymal sperm counts Cauda epididymides were dissected immediately following euthanasia.

Techniques: Labeling, Marker

Journal: Biology of Reproduction

Article Title: Cell-autonomous requirement for mammalian target of rapamycin ( Mtor ) in spermatogonial proliferation and differentiation in the mouse †

doi: 10.1093/biolre/iox022

Figure Lengend Snippet: Only undifferentiated spermatogonia are retained in P18 Mtor KO testes. In each row, representative images of immunofluorescence for specific spermatogonial fate marker proteins (colors indicate the specific marker on each panel), including ZBTB16 (A, B), GFRA1 (C, D), and KIT (E, F) as well as the proliferation marker MKI67 (G, H) along with TRA98 to mark all germ cells. Nuclei are labeled with DAPI, in blue. The accompanying quantitation for WT and KO testes is presented alongside each panel. Asterisks denote statistical significance (**P < 0.01). Error bars indicate one standard deviation. Scale bar (in A) = 50 μm.

Article Snippet: Images for quantitation were acquired with a Fluoview FV1000 laser scanning confocal microscope (Olympus America). table ft1 table-wrap mode="anchored" t5 Table 1. caption a7 Protein Vendor (Catalog Number) Dilution STRA8 Abcam (ab49602) 1:3000 ZBTB16 Santa Cruz (sc-22839) 1:500 c-PARP1 Cell Signaling Technology (#9544) 1:100 DDX4 R&D Systems (AF2030), Abcam (ab13840) 1:800, 1:1000 GATA4 Santa Cruz (sc-1237) 1:100 GFRA1 Proteintech (AF560) 1:800 KIT Cell Signaling Technology (#3074) 1:1000 MTOR Cell Signaling Technology (#2983P) 1:400 p-RPS6 Cell Signaling Technology (#5364P) 1:800 TRA98 Abcam (ab82427) 1:1000 Open in a separate window Antibodies Cauda epididymal sperm counts Cauda epididymides were dissected immediately following euthanasia.

Techniques: Immunofluorescence, Marker, Labeling, Quantitation Assay, Standard Deviation

Journal: Biology of Reproduction

Article Title: Cell-autonomous requirement for mammalian target of rapamycin ( Mtor ) in spermatogonial proliferation and differentiation in the mouse †

doi: 10.1093/biolre/iox022

Figure Lengend Snippet: Spermatogonial differentiation is blocked in Mtor KO mice at P8. In each row, representative images of immunofluorescence for specific spermatogonial fate marker proteins (colors indicate the specific marker on each panel), including ZBTB16 (A, B), GFRA1 (C, D), KIT (E, F), and STRA8 (G, H) along with DDX4 to mark all germ cells (in red, A, B and E–H). Nuclei are labeled (in A–F) with DAPI, in blue. The accompanying quantitation for WT and KO testes is presented alongside each panel. Asterisks denote statistical significance (*P < 0.05, **P < 0.01). Error bars indicate one standard deviation. Scale bar (in A) = 50 μm.

Article Snippet: Images for quantitation were acquired with a Fluoview FV1000 laser scanning confocal microscope (Olympus America). table ft1 table-wrap mode="anchored" t5 Table 1. caption a7 Protein Vendor (Catalog Number) Dilution STRA8 Abcam (ab49602) 1:3000 ZBTB16 Santa Cruz (sc-22839) 1:500 c-PARP1 Cell Signaling Technology (#9544) 1:100 DDX4 R&D Systems (AF2030), Abcam (ab13840) 1:800, 1:1000 GATA4 Santa Cruz (sc-1237) 1:100 GFRA1 Proteintech (AF560) 1:800 KIT Cell Signaling Technology (#3074) 1:1000 MTOR Cell Signaling Technology (#2983P) 1:400 p-RPS6 Cell Signaling Technology (#5364P) 1:800 TRA98 Abcam (ab82427) 1:1000 Open in a separate window Antibodies Cauda epididymal sperm counts Cauda epididymides were dissected immediately following euthanasia.

Techniques: Immunofluorescence, Marker, Labeling, Quantitation Assay, Standard Deviation

Journal: Biology of Reproduction

Article Title: Cell-autonomous requirement for mammalian target of rapamycin ( Mtor ) in spermatogonial proliferation and differentiation in the mouse †

doi: 10.1093/biolre/iox022

Figure Lengend Snippet: Adult Mtor KO spermatogonia incorporate BrdU. Immunostaining was done to localize the pan germ cell marker TRA98 (in red) and the synthetic nucleotide analog BrdU (green) in testes of adult Mtor WT (A) and KO (B) animals. Examples of BrdU+ germ cells are indicated by white arrows and the boundaries of seminiferous tubules are outlined with dashed white lines. Scale bar (in A) = 50 μm.

Article Snippet: Images for quantitation were acquired with a Fluoview FV1000 laser scanning confocal microscope (Olympus America). table ft1 table-wrap mode="anchored" t5 Table 1. caption a7 Protein Vendor (Catalog Number) Dilution STRA8 Abcam (ab49602) 1:3000 ZBTB16 Santa Cruz (sc-22839) 1:500 c-PARP1 Cell Signaling Technology (#9544) 1:100 DDX4 R&D Systems (AF2030), Abcam (ab13840) 1:800, 1:1000 GATA4 Santa Cruz (sc-1237) 1:100 GFRA1 Proteintech (AF560) 1:800 KIT Cell Signaling Technology (#3074) 1:1000 MTOR Cell Signaling Technology (#2983P) 1:400 p-RPS6 Cell Signaling Technology (#5364P) 1:800 TRA98 Abcam (ab82427) 1:1000 Open in a separate window Antibodies Cauda epididymal sperm counts Cauda epididymides were dissected immediately following euthanasia.

Techniques: Immunostaining, Marker

Journal: Biology of Reproduction

Article Title: Cell-autonomous requirement for mammalian target of rapamycin ( Mtor ) in spermatogonial proliferation and differentiation in the mouse †

doi: 10.1093/biolre/iox022

Figure Lengend Snippet: Antibodies

Article Snippet: Images for quantitation were acquired with a Fluoview FV1000 laser scanning confocal microscope (Olympus America). table ft1 table-wrap mode="anchored" t5 Table 1. caption a7 Protein Vendor (Catalog Number) Dilution STRA8 Abcam (ab49602) 1:3000 ZBTB16 Santa Cruz (sc-22839) 1:500 c-PARP1 Cell Signaling Technology (#9544) 1:100 DDX4 R&D Systems (AF2030), Abcam (ab13840) 1:800, 1:1000 GATA4 Santa Cruz (sc-1237) 1:100 GFRA1 Proteintech (AF560) 1:800 KIT Cell Signaling Technology (#3074) 1:1000 MTOR Cell Signaling Technology (#2983P) 1:400 p-RPS6 Cell Signaling Technology (#5364P) 1:800 TRA98 Abcam (ab82427) 1:1000 Open in a separate window Antibodies Cauda epididymal sperm counts Cauda epididymides were dissected immediately following euthanasia.

Techniques:

Journal: Biology of Reproduction

Article Title: The mTORC1 component RPTOR is required for maintenance of the foundational spermatogonial stem cell pool in mice †

doi: 10.1093/biolre/ioy198

Figure Lengend Snippet: Antibodies.

Article Snippet: Images for quantitation were acquired with a Fluoview FV1000 laser scanning confocal microscope (Olympus America). table ft1 table-wrap mode="anchored" t5 Table 1. caption a7 Protein Vendor (Catalog Number) Dilution STRA8 Abcam (ab49602) 1:3000 c-PARP1 Cell Signaling Technology (94 885) 1:100 DDX4 R&D Systems (AF2030), Abcam (ab13840) 1:800, 1:1000 GATA4 Santa Cruz (sc-1237) 1:100 GFRA1 R&D Systems (AF560) 1:800 KIT Cell Signaling Technology (#3074) 1:1000 CRE Cell Signaling Technology (#15 036) 1:100 p-RPS6 Cell Signaling Technology (#5364P) 1:800 TRA98 Abcam (ab82527) 1:1000 H1T Gift from Mary Ann Handel [ 33 ] 1:500 MKI67 Abcam (ab15580) 1:400 SYCP3 Proteintech (#23024–1-AP) 1:250 FOXO1 Cell Signaling Technology (#2880) 1:100 p-AKT Cell Signaling Technology (#4060) 1:200 CDH1 Cell Signaling Technology (#3195) 1:200 Open in a separate window Antibodies.

Techniques:

Journal: Biology of Reproduction

Article Title: The mTORC1 component RPTOR is required for maintenance of the foundational spermatogonial stem cell pool in mice †

doi: 10.1093/biolre/ioy198

Figure Lengend Snippet: P8 Rptor KO testes have reduced germ cell numbers but normal percentages of undifferentiated and differentiating spermatogonia. In each row, representative images of immunostaining for specific proteins and accompanying quantitation are shown for P8 WT and KO testes. (A, B) The entire germ cell population is DDX4+ (green), and nuclei are stained with DAPI (blue). (C, D) Undifferentiated stem and progenitor spermatogonia are GFRA1+ (green), all germ cells DDX4+ (red), and nuclei are stained with DAPI (blue). (E, F) Differentiating spermatogonia are KIT+ (green), all germ cells are TRA98+ (red), and nuclei are stained with DAPI (blue). (G, H) Germ cells responding to retinoic acid are STRA8+ (green), all germ cells are DDX4+ (red), and nuclei are stained with DAPI (blue). (I, J) Germ cells staining positive for the meiotic marker SYCP3 (green), all germ cells are TRA98+ (red). Error bars indicate one standard deviation, and asterisks denote statistical significance at P < 0.05. Scale bar (in A) = 50 μm.

Article Snippet: Images for quantitation were acquired with a Fluoview FV1000 laser scanning confocal microscope (Olympus America). table ft1 table-wrap mode="anchored" t5 Table 1. caption a7 Protein Vendor (Catalog Number) Dilution STRA8 Abcam (ab49602) 1:3000 c-PARP1 Cell Signaling Technology (94 885) 1:100 DDX4 R&D Systems (AF2030), Abcam (ab13840) 1:800, 1:1000 GATA4 Santa Cruz (sc-1237) 1:100 GFRA1 R&D Systems (AF560) 1:800 KIT Cell Signaling Technology (#3074) 1:1000 CRE Cell Signaling Technology (#15 036) 1:100 p-RPS6 Cell Signaling Technology (#5364P) 1:800 TRA98 Abcam (ab82527) 1:1000 H1T Gift from Mary Ann Handel [ 33 ] 1:500 MKI67 Abcam (ab15580) 1:400 SYCP3 Proteintech (#23024–1-AP) 1:250 FOXO1 Cell Signaling Technology (#2880) 1:100 p-AKT Cell Signaling Technology (#4060) 1:200 CDH1 Cell Signaling Technology (#3195) 1:200 Open in a separate window Antibodies.

Techniques: Immunostaining, Quantitation Assay, Staining, Marker, Standard Deviation

Journal: Biology of Reproduction

Article Title: The mTORC1 component RPTOR is required for maintenance of the foundational spermatogonial stem cell pool in mice †

doi: 10.1093/biolre/ioy198

Figure Lengend Snippet: Antibodies.

Article Snippet: Images for quantitation were acquired with a Fluoview FV1000 laser scanning confocal microscope (Olympus America). table ft1 table-wrap mode="anchored" t5 Table 1. caption a7 Protein Vendor (Catalog Number) Dilution STRA8 Abcam (ab49602) 1:3000 c-PARP1 Cell Signaling Technology (94 885) 1:100 DDX4 R&D Systems (AF2030), Abcam (ab13840) 1:800, 1:1000 GATA4 Santa Cruz (sc-1237) 1:100 GFRA1 R&D Systems (AF560) 1:800 KIT Cell Signaling Technology (#3074) 1:1000 CRE Cell Signaling Technology (#15 036) 1:100 p-RPS6 Cell Signaling Technology (#5364P) 1:800 TRA98 Abcam (ab82527) 1:1000 H1T Gift from Mary Ann Handel [ 33 ] 1:500 MKI67 Abcam (ab15580) 1:400 SYCP3 Proteintech (#23024–1-AP) 1:250 FOXO1 Cell Signaling Technology (#2880) 1:100 p-AKT Cell Signaling Technology (#4060) 1:200 CDH1 Cell Signaling Technology (#3195) 1:200 Open in a separate window Antibodies.

Techniques:

Journal: Biology of Reproduction

Article Title: The mTORC1 component RPTOR is required for maintenance of the foundational spermatogonial stem cell pool in mice †

doi: 10.1093/biolre/ioy198

Figure Lengend Snippet: Antibodies.

Article Snippet: Images for quantitation were acquired with a Fluoview FV1000 laser scanning confocal microscope (Olympus America). table ft1 table-wrap mode="anchored" t5 Table 1. caption a7 Protein Vendor (Catalog Number) Dilution STRA8 Abcam (ab49602) 1:3000 c-PARP1 Cell Signaling Technology (94 885) 1:100 DDX4 R&D Systems (AF2030), Abcam (ab13840) 1:800, 1:1000 GATA4 Santa Cruz (sc-1237) 1:100 GFRA1 R&D Systems (AF560) 1:800 KIT Cell Signaling Technology (#3074) 1:1000 CRE Cell Signaling Technology (#15 036) 1:100 p-RPS6 Cell Signaling Technology (#5364P) 1:800 TRA98 Abcam (ab82527) 1:1000 H1T Gift from Mary Ann Handel [ 33 ] 1:500 MKI67 Abcam (ab15580) 1:400 SYCP3 Proteintech (#23024–1-AP) 1:250 FOXO1 Cell Signaling Technology (#2880) 1:100 p-AKT Cell Signaling Technology (#4060) 1:200 CDH1 Cell Signaling Technology (#3195) 1:200 Open in a separate window Antibodies.

Techniques:

Journal: International journal of molecular sciences

Article Title: Repetitive Low-Level Blast Exposure via Akt/NF-κB Signaling Pathway Mediates the M1 Polarization of Mouse Alveolar Macrophage MH-S Cells.

doi: 10.3390/ijms241310596

Figure Lengend Snippet: Figure 2. Effect of rLLB exposure on morphological and phenotype transformation of MH-S cells. (a) Optical microscope images of MH-S cells in NC and rLLB group at 6h. The right panel (scale bar: 25 µm) is a local magnification of the left panel (scale bar: 100 µm). (b,c) The statistics of the cell morphology change rate and cell aspect ratio in the graphs of (a) (n = 10). (d) Confocal microscopy images of cells in NC and rLLB groups stained for nuclei (DAPI, blue), β-Tubulin (Red), and Phalloidin (green) to detect cytoskeletal changes (scale bars: 20 µm). (e–g,i) qPCR and WB analysis of the expression of M1 biomarkers iNOS, CD86, IL-6 and M2 biomarkers Arg1, CD206, IL-10 at 6 h, 12 h, 24 h, 36 h, 48 h in NC and rLLB groups. (h,j) Quantification of the grayscale values of (g,i), respectively (n = 3). (k,m) Immunofluorescence analysis of the iNOS and Arg1 expression in MH-S cells at 24 h (scale bar: 100 µm). (l,n) Quantification of the mean fluorescence intensity (MFI) of iNOS and Arg1 expression at 12 h, 24 h, and 36 h, respectively (n = 3). Data are presented as the means ± SD. The NC groups at different time points in (e,f,h,j) are normalized to 1, respectively. (b,c) Using unpaired t test. (e,f,h,j,l,n) Using two-way ANOVA followed by Sidak’s multiple comparisons test. * p < 0.05, ** p < 0.01, *** p < 0.001, ns: no significance.

Article Snippet: After blocking with 5% skimmed milk, the membranes were probed with primary antibodies against iNOS (1:1000, # AF0199, Affinity, Liyang, Jiangsu, China), CD86 (1:500, #sc-28347, Santa Cruz, Santa Cruz, CA, USA), IL-6 (1:500, #sc-57345, Santa Cruz, CA, USA), Arg1 (1:5000, #16001-1-AP, Proteintech, China), CD206 (1:500, #sc-58986, Santa Cruz, CA, USA), IL-10 (1:500, #sc-365858, Santa Cruz, USA), Bax (1:1000, #AF0120, Affinity, Liyang, China), Bcl2 (1:1000, #AF6139, Affinity, Liyang, China), Caspase3 (1:1000, #AF6311, Affinity, Liyang, China), Cleaved-caspase3 (1:1000, #AF7022, Affinity, Liyang, China), Akt (1:1000, #AF6261, Affinity, Liyang, China), p-Akt (Ser473) (1:1000, #AF0016, Affinity, Liyang, China), NF-κB p105/p50 (1:1000, #AF6217, Affinity, China), NF-κB p-p105/p50 (Ser337) (1:1000, #AF3219, Affinity, Liyang, China), NF-κB p65 (1:1000, #AF5006, Affinity, Liyang, China), NF-κB p-p65 (Ser536) (1:1000, #AF2006, Affinity, China), GAPDH (1:5000, K200103M, Solarbio, Beijing, China) and β-Tubulin (1:5000, #KM9003T, SUNGENE, China) at 4 ◦C overnight.

Techniques: Transformation Assay, Microscopy, Confocal Microscopy, Staining, Expressing, Immunofluorescence, Fluorescence

Journal: International journal of molecular sciences

Article Title: Repetitive Low-Level Blast Exposure via Akt/NF-κB Signaling Pathway Mediates the M1 Polarization of Mouse Alveolar Macrophage MH-S Cells.

doi: 10.3390/ijms241310596

Figure Lengend Snippet: Figure 5. Effect of GDC-0068 on the phenotype transformation of MH-S cells induced by rLLB exposure. (a–c) qPCR and WB analysis of the effect of adding GDC-0068 (10 µM) on iNOS, CD86, IL-6, Arg1, CD206, and IL-10 expression in MH-S cells at 24 h. (d,e) Quantification of the grayscale values of (c). (f,h) Flow cytometry analysis of the effect of adding GDC-0068 (10 µM) on the ratio of M1/M2 macrophages at 24 h by CD86 and CD206 labeling. (g,i) Quantification of the CD86 or CD206 positive cells proportion of (f,h), respectively. (j,k) Immunofluorescence analysis of the effect of adding GDC-0068 (10 µM) on the expression of IL-6 and IL-10 in MH-S cells at 24 h. (l,m) Quantification of MFI in (j,k), respectively. Data are presented as the means ± SD (n = 3). All statistics in this figure use one-way ANOVA followed by Tukey’s multiple comparisons tests. * p < 0.05, ** p < 0.01, *** p < 0.001, ns: no significance.

Article Snippet: After blocking with 5% skimmed milk, the membranes were probed with primary antibodies against iNOS (1:1000, # AF0199, Affinity, Liyang, Jiangsu, China), CD86 (1:500, #sc-28347, Santa Cruz, Santa Cruz, CA, USA), IL-6 (1:500, #sc-57345, Santa Cruz, CA, USA), Arg1 (1:5000, #16001-1-AP, Proteintech, China), CD206 (1:500, #sc-58986, Santa Cruz, CA, USA), IL-10 (1:500, #sc-365858, Santa Cruz, USA), Bax (1:1000, #AF0120, Affinity, Liyang, China), Bcl2 (1:1000, #AF6139, Affinity, Liyang, China), Caspase3 (1:1000, #AF6311, Affinity, Liyang, China), Cleaved-caspase3 (1:1000, #AF7022, Affinity, Liyang, China), Akt (1:1000, #AF6261, Affinity, Liyang, China), p-Akt (Ser473) (1:1000, #AF0016, Affinity, Liyang, China), NF-κB p105/p50 (1:1000, #AF6217, Affinity, China), NF-κB p-p105/p50 (Ser337) (1:1000, #AF3219, Affinity, Liyang, China), NF-κB p65 (1:1000, #AF5006, Affinity, Liyang, China), NF-κB p-p65 (Ser536) (1:1000, #AF2006, Affinity, China), GAPDH (1:5000, K200103M, Solarbio, Beijing, China) and β-Tubulin (1:5000, #KM9003T, SUNGENE, China) at 4 ◦C overnight.

Techniques: Transformation Assay, Expressing, Flow Cytometry, Labeling, Immunofluorescence

Journal: International journal of molecular sciences

Article Title: Repetitive Low-Level Blast Exposure via Akt/NF-κB Signaling Pathway Mediates the M1 Polarization of Mouse Alveolar Macrophage MH-S Cells.

doi: 10.3390/ijms241310596

Figure Lengend Snippet: Figure 6. Effect of PDTC on the phenotype transformation of MH-S cells induced by rLLB exposure. (a–c) qPCR and WB analysis of the effect of adding PDTC (20 µM) on iNOS, CD86, IL-6, Arg1, CD206, and IL-10 expression in MH-S cells at 24 h. (d,e) Quantification of the grayscale values of (c). Data are presented as the means ± SD (n = 3). All statistics in this figure use one-way ANOVA followed by Tukey’s multiple comparisons tests. * p < 0.05, ** p < 0.01, *** p < 0.001, ns: no significance.

Article Snippet: After blocking with 5% skimmed milk, the membranes were probed with primary antibodies against iNOS (1:1000, # AF0199, Affinity, Liyang, Jiangsu, China), CD86 (1:500, #sc-28347, Santa Cruz, Santa Cruz, CA, USA), IL-6 (1:500, #sc-57345, Santa Cruz, CA, USA), Arg1 (1:5000, #16001-1-AP, Proteintech, China), CD206 (1:500, #sc-58986, Santa Cruz, CA, USA), IL-10 (1:500, #sc-365858, Santa Cruz, USA), Bax (1:1000, #AF0120, Affinity, Liyang, China), Bcl2 (1:1000, #AF6139, Affinity, Liyang, China), Caspase3 (1:1000, #AF6311, Affinity, Liyang, China), Cleaved-caspase3 (1:1000, #AF7022, Affinity, Liyang, China), Akt (1:1000, #AF6261, Affinity, Liyang, China), p-Akt (Ser473) (1:1000, #AF0016, Affinity, Liyang, China), NF-κB p105/p50 (1:1000, #AF6217, Affinity, China), NF-κB p-p105/p50 (Ser337) (1:1000, #AF3219, Affinity, Liyang, China), NF-κB p65 (1:1000, #AF5006, Affinity, Liyang, China), NF-κB p-p65 (Ser536) (1:1000, #AF2006, Affinity, China), GAPDH (1:5000, K200103M, Solarbio, Beijing, China) and β-Tubulin (1:5000, #KM9003T, SUNGENE, China) at 4 ◦C overnight.

Techniques: Transformation Assay, Expressing