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Antibodies Used for Different Experiments in This Study

Journal: Endocrinology

Article Title: Myosin VIIa Supports Spermatid/Organelle Transport and Cell Adhesion During Spermatogenesis in the Rat Testis

doi: 10.1210/en.2018-00855

Figure Lengend Snippet: Antibodies Used for Different Experiments in This Study

Article Snippet: Working Dilution IB Analysis IF Analysis Myosin VIIA (RRID: AB_2148626 ) ( 34 ) Mouse Santa Cruz Biotechnology sc-74516 1:200 1:100 Myosin VIIA (RRID: AB_303841 ) ( 35 ) Rabbit Abcam ab3481 1:5000 1:400 EB1 (RRID: AB_397891 ) ( 36 ) Mouse BD Biosciences 610534 — 1:200 ZO-1 (RRID: AB_2533938 ) ( 37 ) Rabbit Invitrogen 61-7300 1:250 1:100 Occludin (RRID: AB_2533977 ) ( 38 ) Rabbit Invitrogen 71-1500 1:250 1:100 CAR (RRID: AB_2087557 ) ( 39 ) Rabbit Santa Cruz Biotechnology sc-15405 1:200 1:50 JAM-A (RRID: AB_2533241 ) ( 40 ) Rabbit Invitrogen 36-1700 1:250 — Claudin 11 (RRID: AB_2533259 ) ( 41 ) Rabbit Invitrogen 36-4500 1:250 — N-cadherin (RRID: AB_2313779 ) ( 42 ) Mouse Invitrogen 33-3900 — 1:100 N-cadherin (RRID: AB_647794 ) ( 43 ) Rabbit Santa Cruz Biotechnology sc-7939 1:200 — β -Catenin (RRID: AB_2533982 ) ( 44 ) Rabbit Invitrogen 71-2700 1:250 1:100 Arp3 (RRID: AB_476749 ) ( 45 ) Mouse Sigma-Aldrich A5979 1:3000 1:50 Eps8 (RRID: AB_397544 ) ( 46 ) Mouse Invitrogen 610143 1:5000 1:50 N -WASP (RRID: AB_2288632 ) ( 47 ) Rabbit Santa Cruz Biotechnology sc-20770 1:200 — Plastin 3 (RRID: AB_2636816 ) ( 48 ) Rabbit Abcam ab137585 1:500 — β 1 -Integrin (RRID: AB_2130101 ) ( 49 ) Rabbit Santa Cruz Biotechnology sc-8978 — 1:50 α -Tubulin (RRID: AB_2241126 ) ( 50 ) Mouse Abcam ab7291 — 1:200 Detyrosinated α -tubulin (RRID: AB_869990 ) ( 51 ) Rabbit Abcam ab48389 — 1:200 β -Tubulin (RRID: AB_2210370 ) ( 52 ) Rabbit Abcam ab6046 1:5000 1:200 Tyr-tubulin (RRID: AB_305328 ) ( 53 ) Rat Abcam ab6160 — 1:200 Acetylated α -tubulin (RRID: AB_448182 ) ( 54 ) Mouse Abcam ab24610 — 1:200 β -Actin (RRID: AB_630836 ) ( 55 ) Goat Santa Cruz Biotechnology sc-1616 1:200 — Vimentin (RRID: AB_628437 ) ( 56 ) Mouse Santa Cruz Biotechnology sc-6260 1:200 1:200 Gapdh (RRID: AB_2107448 ) ( 57 ) Mouse Abcam ab8245 1:1000 — Laminin γ 3 (RRID: AB_2636817 ) ( 58 ) Rabbit Cheng Laboratory ( 67 ) — — 1:200 Goat IgG-HRP (RRID: AB_634811 ) ( 59 ) Bovine Santa Cruz Biotechnology sc-2350 1:3000 — Rabbit IgG-HRP (RRID: AB_634837 ) ( 60 ) Bovine Santa Cruz Biotechnology sc-2370 1:3000 — Mouse IgG-HRP (RRID: AB_634824 ) ( 61 ) Bovine Santa Cruz Biotechnology sc-2371 1:3000 — Rabbit IgG–Alexa Fluor 488 (RRID: AB_2576217 ) ( 62 ) Goat Invitrogen A-11034 — 1:250 Rabbit IgG–Alexa Fluor 555 (RRID: AB_2535850 ) ( 63 ) Goat Invitrogen A-21429 — 1:250 Mouse IgG–Alexa Fluor 488 (RRID: AB_2534088 ) ( 64 ) Goat Invitrogen A-11029 — 1:250 Mouse IgG–Alexa Fluor 555 (RRID: AB_141780 ) ( 65 ) Goat Invitrogen A-21424 — 1:250 Rat IgG–Alexa Fluor 488 (RRID: AB_141373 ) ( 66 ) Goat Invitrogen A-11006 — 1:250 Open in a separate window Antibodies used herein cross-reacted with the corresponding proteins in rats.

Techniques:

Myosin VIIa knockdown-induced disorganization of F-actin at the ES is mediated through changes in the spatiotemporal expression of actin regulatory proteins Arp3 and Eps8. In this study, we examined whether there were changes in the spatial expression of two actin regulatory proteins, Arp3 and Eps8. First, the efficacy of myosin VIIa knockdown was confirmed by IF analysis of myosin VIIa in the epithelium. Myosin VIIa knockdown in the testis reduced myosin VIIa expression at the apical and basal ES in the testis by as much as ∼70% (see also bar graph in the right panel). In stage VII tubules of control testes, Arp3 and Eps8 were highly expressed at the concave side of spermatid heads, appearing as bulblike structures to support extensive endocytic protein trafficking events at the site; both proteins were also expressed at the basal ES/BTB near the basement membrane (annotated by the dashed white line). However, after myosin VIIa knockdown, both Arp3 and Eps8 were considerably downregulated and mislocalized at the apical ES, and many elongated spermatids displayed defects in their polarity (yellow arrowheads). These changes thus impeded the localization of a spermatid-specific apical ES adhesion protein laminin γ3 chain (67, 83), because laminin γ3 was no longer highly expressed at the tip of elongated spermatids, failing to support spermatid adhesion. The bar graph in the right panel also illustrates a considerable reduction in the percentage of stage VII tubules and an increase in the percentage of stage VIII tubules due to defects in spermiation after myosin VIIa knockdown. This is due to the release of sperms that took place in stage VII tubules, thus making these tubules similar to stage VIII tubules when tubules were randomly scored. Scale bars, 80 µm; 40 µm in insets that are magnified images boxed in green or yellow.

Journal: Endocrinology

Article Title: Myosin VIIa Supports Spermatid/Organelle Transport and Cell Adhesion During Spermatogenesis in the Rat Testis

doi: 10.1210/en.2018-00855

Figure Lengend Snippet: Myosin VIIa knockdown-induced disorganization of F-actin at the ES is mediated through changes in the spatiotemporal expression of actin regulatory proteins Arp3 and Eps8. In this study, we examined whether there were changes in the spatial expression of two actin regulatory proteins, Arp3 and Eps8. First, the efficacy of myosin VIIa knockdown was confirmed by IF analysis of myosin VIIa in the epithelium. Myosin VIIa knockdown in the testis reduced myosin VIIa expression at the apical and basal ES in the testis by as much as ∼70% (see also bar graph in the right panel). In stage VII tubules of control testes, Arp3 and Eps8 were highly expressed at the concave side of spermatid heads, appearing as bulblike structures to support extensive endocytic protein trafficking events at the site; both proteins were also expressed at the basal ES/BTB near the basement membrane (annotated by the dashed white line). However, after myosin VIIa knockdown, both Arp3 and Eps8 were considerably downregulated and mislocalized at the apical ES, and many elongated spermatids displayed defects in their polarity (yellow arrowheads). These changes thus impeded the localization of a spermatid-specific apical ES adhesion protein laminin γ3 chain (67, 83), because laminin γ3 was no longer highly expressed at the tip of elongated spermatids, failing to support spermatid adhesion. The bar graph in the right panel also illustrates a considerable reduction in the percentage of stage VII tubules and an increase in the percentage of stage VIII tubules due to defects in spermiation after myosin VIIa knockdown. This is due to the release of sperms that took place in stage VII tubules, thus making these tubules similar to stage VIII tubules when tubules were randomly scored. Scale bars, 80 µm; 40 µm in insets that are magnified images boxed in green or yellow.

Article Snippet: Working Dilution IB Analysis IF Analysis Myosin VIIA (RRID: AB_2148626 ) ( 34 ) Mouse Santa Cruz Biotechnology sc-74516 1:200 1:100 Myosin VIIA (RRID: AB_303841 ) ( 35 ) Rabbit Abcam ab3481 1:5000 1:400 EB1 (RRID: AB_397891 ) ( 36 ) Mouse BD Biosciences 610534 — 1:200 ZO-1 (RRID: AB_2533938 ) ( 37 ) Rabbit Invitrogen 61-7300 1:250 1:100 Occludin (RRID: AB_2533977 ) ( 38 ) Rabbit Invitrogen 71-1500 1:250 1:100 CAR (RRID: AB_2087557 ) ( 39 ) Rabbit Santa Cruz Biotechnology sc-15405 1:200 1:50 JAM-A (RRID: AB_2533241 ) ( 40 ) Rabbit Invitrogen 36-1700 1:250 — Claudin 11 (RRID: AB_2533259 ) ( 41 ) Rabbit Invitrogen 36-4500 1:250 — N-cadherin (RRID: AB_2313779 ) ( 42 ) Mouse Invitrogen 33-3900 — 1:100 N-cadherin (RRID: AB_647794 ) ( 43 ) Rabbit Santa Cruz Biotechnology sc-7939 1:200 — β -Catenin (RRID: AB_2533982 ) ( 44 ) Rabbit Invitrogen 71-2700 1:250 1:100 Arp3 (RRID: AB_476749 ) ( 45 ) Mouse Sigma-Aldrich A5979 1:3000 1:50 Eps8 (RRID: AB_397544 ) ( 46 ) Mouse Invitrogen 610143 1:5000 1:50 N -WASP (RRID: AB_2288632 ) ( 47 ) Rabbit Santa Cruz Biotechnology sc-20770 1:200 — Plastin 3 (RRID: AB_2636816 ) ( 48 ) Rabbit Abcam ab137585 1:500 — β 1 -Integrin (RRID: AB_2130101 ) ( 49 ) Rabbit Santa Cruz Biotechnology sc-8978 — 1:50 α -Tubulin (RRID: AB_2241126 ) ( 50 ) Mouse Abcam ab7291 — 1:200 Detyrosinated α -tubulin (RRID: AB_869990 ) ( 51 ) Rabbit Abcam ab48389 — 1:200 β -Tubulin (RRID: AB_2210370 ) ( 52 ) Rabbit Abcam ab6046 1:5000 1:200 Tyr-tubulin (RRID: AB_305328 ) ( 53 ) Rat Abcam ab6160 — 1:200 Acetylated α -tubulin (RRID: AB_448182 ) ( 54 ) Mouse Abcam ab24610 — 1:200 β -Actin (RRID: AB_630836 ) ( 55 ) Goat Santa Cruz Biotechnology sc-1616 1:200 — Vimentin (RRID: AB_628437 ) ( 56 ) Mouse Santa Cruz Biotechnology sc-6260 1:200 1:200 Gapdh (RRID: AB_2107448 ) ( 57 ) Mouse Abcam ab8245 1:1000 — Laminin γ 3 (RRID: AB_2636817 ) ( 58 ) Rabbit Cheng Laboratory ( 67 ) — — 1:200 Goat IgG-HRP (RRID: AB_634811 ) ( 59 ) Bovine Santa Cruz Biotechnology sc-2350 1:3000 — Rabbit IgG-HRP (RRID: AB_634837 ) ( 60 ) Bovine Santa Cruz Biotechnology sc-2370 1:3000 — Mouse IgG-HRP (RRID: AB_634824 ) ( 61 ) Bovine Santa Cruz Biotechnology sc-2371 1:3000 — Rabbit IgG–Alexa Fluor 488 (RRID: AB_2576217 ) ( 62 ) Goat Invitrogen A-11034 — 1:250 Rabbit IgG–Alexa Fluor 555 (RRID: AB_2535850 ) ( 63 ) Goat Invitrogen A-21429 — 1:250 Mouse IgG–Alexa Fluor 488 (RRID: AB_2534088 ) ( 64 ) Goat Invitrogen A-11029 — 1:250 Mouse IgG–Alexa Fluor 555 (RRID: AB_141780 ) ( 65 ) Goat Invitrogen A-21424 — 1:250 Rat IgG–Alexa Fluor 488 (RRID: AB_141373 ) ( 66 ) Goat Invitrogen A-11006 — 1:250 Open in a separate window Antibodies used herein cross-reacted with the corresponding proteins in rats.

Techniques: Expressing

Mycobacterium species used in this study and the results of the ASL cytopathicity assays for the slow-growing mycobacteria a

Journal:

Article Title: Analysis of Mycobacterium Species for the Presence of a Macrolide Toxin, Mycolactone

doi: 10.1128/IAI.72.1.123-132.2004

Figure Lengend Snippet: Mycobacterium species used in this study and the results of the ASL cytopathicity assays for the slow-growing mycobacteria a

Article Snippet: 1306, M. scrofulaceum (ATCC 35787) , TMCC , 10,000.

Techniques: