polyclonal anti-actl7b  (Thermo Fisher)

Journal: Development (Cambridge, England)

Article Title: Actl7b deficiency leads to mislocalization of LC8 type dynein light chains and disruption of murine spermatogenesis

doi: 10.1242/dev.201593

Figure Lengend Snippet: Establishment of Actl7b -deficient mice. (A) Graphical representation of CRISPR-Cas9-mediated gene editing of the Actl7b locus using two guide RNAs (black arrowheads) targeting the intron-less Actl7b -coding sequence. 473 bp were deleted, causing a frameshift leading to a premature stop. (B) Agarose gel of genotyping polymerase chain reaction of Actl7b +/+ , Actl7b +/− and Actl7b −/− mice (wild-type band, 607 bp; KO band, 134 bp). (C) Actl7b expression and ACTL7B immunolocalization during spermiogenesis based on literature ( ; ; ). ES, elongating spermatids; P, pachytene spermatocytes; RS, round spermatids; S, spermoatogonia; SP, spermatozoa. (D) Immunohistochemical staining against ACTL7B on Bouin-fixed, paraffin wax-embedded Actl7b +/+ , Actl7b +/− and Actl7b −/− testis sections counterstained with Hematoxylin. Scale bars: 20 μm.

Article Snippet: Protein lysates of the five clones were tested by western blotting using polyclonal anti-ACTL7B (Invitrogen, PA5-113560, 1:1000).

Techniques: CRISPR, Sequencing, Agarose Gel Electrophoresis, Polymerase Chain Reaction, Expressing, Immunohistochemistry, Staining, Paraffin Wax

Journal: Development (Cambridge, England)

Article Title: Actl7b deficiency leads to mislocalization of LC8 type dynein light chains and disruption of murine spermatogenesis

doi: 10.1242/dev.201593

Figure Lengend Snippet: Fertility analysis and reproductive organ morphology. (A) Pregnancy rate of Actl7b +/+ , Actl7b +/− and Actl7b −/− males mated with female wild-type C57BL/6J mice ( n =number of males). (B) Average litter sizes monitored after mating of Actl7b +/+ and Actl7b +/− males with female wild-type C57BL/6J mice ( n =number of males). Five plugs per male were recorded. (C) Mean testis weight of Actl7b +/+ , Actl7b +/− and Actl7b −/− males ( n =number of males). (D) Testis to body weight ratio of Actl7b +/+ , Actl7b +/− and Actl7b −/− males ( n =number of males). (E) Photographs of representative testes dissected from Actl7b +/+ , Actl7b +/− and Actl7b −/− littermates with similar body weight. (F) Hematoxylin and Eosin staining of testis (stages IV-VI of the epithelial cycle), caput epididymis and cauda epididymis of Actl7b +/+ , Actl7b +/− and Actl7b −/− males. Scale bars: 50 μm. Statistical analyses were carried out using a two-tailed, unpaired Student's t -test (*** P <0.001). Error bars represent s.d.

Article Snippet: Protein lysates of the five clones were tested by western blotting using polyclonal anti-ACTL7B (Invitrogen, PA5-113560, 1:1000).

Techniques: Staining, Two Tailed Test

Journal: Development (Cambridge, England)

Article Title: Actl7b deficiency leads to mislocalization of LC8 type dynein light chains and disruption of murine spermatogenesis

doi: 10.1242/dev.201593

Figure Lengend Snippet: Morphology of Actl7b -deficient seminiferous tubules. (A) Hematoxylin and Eosin staining of Bouin-fixed paraffin wax-embedded testis sections of Actl7b −/− mice. Immature apoptotic germ cells can be seen to be released into the lumen (green arrows). At late stage VIII, elongated spermatids with an abnormal morphology, which were not spermiated, were seen (green arrowheads) and round spermatids blocked in development with dark cytoplasm were found (orange arrows). Vacuolation of seminiferous tubules was detected (orange arrowheads). Scale bars: 50 µm. (B,C) Transmission electron micrographs of vesicles filled with degrading spermatids detected in Actl7b −/− seminiferous tubules. N, condensed nuclei; dN, degraded nucleus; G, granular material. Orange arrowheads indicate acrosomal structures; green arrowheads indicate flagellar cross-sections; green arrows indicate mitochondria. Scale bars: 5 µm in B; 2 µm in C. (D) Western blots on protein extractions from whole Actl7b +/+ , Actl7b +/− and Actl7b −/− testis. Anti-LC3 and anti-CSTB were used. α-Tubulin was used as a loading control.

Article Snippet: Protein lysates of the five clones were tested by western blotting using polyclonal anti-ACTL7B (Invitrogen, PA5-113560, 1:1000).

Techniques: Staining, Paraffin Wax, Transmission Assay, Western Blot

Journal: Development (Cambridge, England)

Article Title: Actl7b deficiency leads to mislocalization of LC8 type dynein light chains and disruption of murine spermatogenesis

doi: 10.1242/dev.201593

Figure Lengend Snippet: Acrosome formation, flagella formation and chromatin condensation in Actl7b -deficient mice. (A) PNA staining of testis of Actl7b +/+ , Actl7b +/− and Actl7b −/− males. Acrosomal structures in Golgi, cap and maturation phases are shown. Scale bars: 10 μm. (B) Immunohistochemistry staining against ODF2 on Actl7b +/+ (top) Actl7b +/− (middle) and Actl7b −/− (bottom) testis tissue sections. DAPI was used as the counterstain. Scale bars: 10 μm. (C) Immunohistochemistry staining against PRM2 on Actl7b +/+ , Actl7b +/− and Actl7b −/− testis tissue sections. DAPI (in gray) was used as the counterstain. Scale bars: 50 μm.

Article Snippet: Protein lysates of the five clones were tested by western blotting using polyclonal anti-ACTL7B (Invitrogen, PA5-113560, 1:1000).

Techniques: Staining, Immunohistochemistry

Journal: Development (Cambridge, England)

Article Title: Actl7b deficiency leads to mislocalization of LC8 type dynein light chains and disruption of murine spermatogenesis

doi: 10.1242/dev.201593

Figure Lengend Snippet: Ultrastructural analysis of Actl7b −/− testis. (A) Transmission electron micrograph of a lumen of an Actl7b −/− seminiferous tubule. Scale bar: 5 µm. (B) Transmission electron micrograph of a lumen of an Actl7b +/+ seminiferous tubule. Scale bar: 5 µm. (C) Immunohistochemistry against cP2 on caput sections from Actl7b +/+ , Actl7b +/− and Actl7b −/− mice. DAPI was used as a counterstain. Scale bars: 100 µm. (D-G) Images of representative Actl7b −/− spermatids with condensed nuclei. Scale bars: 2 µm.

Article Snippet: Protein lysates of the five clones were tested by western blotting using polyclonal anti-ACTL7B (Invitrogen, PA5-113560, 1:1000).

Techniques: Transmission Assay, Immunohistochemistry

Journal: Development (Cambridge, England)

Article Title: Actl7b deficiency leads to mislocalization of LC8 type dynein light chains and disruption of murine spermatogenesis

doi: 10.1242/dev.201593

Figure Lengend Snippet: Interaction of ACTL7B with DYNLL1 and DYNLL2. Western blots of the protein input (whole wild-type testis), the IP eluate of the anti-DYNLL1-coupled beads (IP D1), the IP eluate of the anti-DYNLL2-coupled beads (IP D2) and the IP eluate of the beads-only control (IP c.). Anti-ACTL7B and anti-DYNLL2 antibodies were used for western blots.

Article Snippet: Protein lysates of the five clones were tested by western blotting using polyclonal anti-ACTL7B (Invitrogen, PA5-113560, 1:1000).

Techniques: Western Blot

Journal: Development (Cambridge, England)

Article Title: Actl7b deficiency leads to mislocalization of LC8 type dynein light chains and disruption of murine spermatogenesis

doi: 10.1242/dev.201593

Figure Lengend Snippet: Localization of DYNLL1 and DYNLL2 in Actl7b -deficient testis. (A) Graphical depiction of DYNLL1 and DYNLL2 immunolocalization during spermiogenesis based on literature . (B) Western blots on protein extracts from whole Actl7b +/+ , Actl7b +/− and Actl7b −/− testis. Anti-ACTL7B, anti-DYNLL2 and anti-DYNLL2 were used. α-Tubulin was used as loading control. (C) DYNLL1 staining in Actl7b +/+ , Actl7b +/− and Actl7b −/− elongating spermatids. DAPI was used as a counterstain. Scale bar: 20 µm. (D) DYNLL2 staining in Actl7b +/+ , Actl7b +/− and Actl7b −/− elongating spermatids. DAPI was used as a counterstain. Scale bar: 20 µm. (E) Immunocytochemical staining against DYNLL2 in wild-type and ACTL7B-eGFP-expressing HEK cells. Scale bars: 50 µm. (F) Immunocytochemical staining against DYNLL1 in wild-type and ACTL7B-eGFP-expressing HEK cells. Scale bars: 50 µm. Images shown in E and F are taken from the overviews displayed in Figs S11 and S12 .

Article Snippet: Protein lysates of the five clones were tested by western blotting using polyclonal anti-ACTL7B (Invitrogen, PA5-113560, 1:1000).

Techniques: Western Blot, Staining, Expressing

Journal: Development (Cambridge, England)

Article Title: Actl7b deficiency leads to mislocalization of LC8 type dynein light chains and disruption of murine spermatogenesis

doi: 10.1242/dev.201593

Figure Lengend Snippet: Proteomic analysis of Actl7b -deficient testis. (A-C) Volcano plots showing differential abundance (DA) of proteins in Actl7b +/− compared with Actl7b +/+ (A), in Actl7b −/− compared with Actl7b +/− (B) and in Actl7b −/− compared with Actl7b +/+ (C) testis. Proteins showing a significant DA are indicated in teal (less abundant) and yellow (more abundant) (adjusted P -value>0.05). Top DA proteins are labeled with their corresponding gene symbol. (D,E) Venn diagrams showing the overlap of significantly higher (D) and lower (E) abundance proteins in the comparisons of Actl7b −/− with Actl7b +/+ , and Actl7b −/− with Actl7b +/+ testis (adjusted P -value≤0.05, LFC≤1).

Article Snippet: Protein lysates of the five clones were tested by western blotting using polyclonal anti-ACTL7B (Invitrogen, PA5-113560, 1:1000).

Techniques: Labeling

Journal: Development (Cambridge, England)

Article Title: Actl7b deficiency leads to mislocalization of LC8 type dynein light chains and disruption of murine spermatogenesis

doi: 10.1242/dev.201593

Figure Lengend Snippet: Evolutionary analysis of ACTL7A and ACTL7B

Article Snippet: Protein lysates of the five clones were tested by western blotting using polyclonal anti-ACTL7B (Invitrogen, PA5-113560, 1:1000).

Techniques:

Journal: PLoS ONE

Article Title: Mouse Tafazzin Is Required for Male Germ Cell Meiosis and Spermatogenesis

doi: 10.1371/journal.pone.0131066

Figure Lengend Snippet: Immunohistology sections of SV129P2 wild type (A,B,E,F,G) and TAZ Neo testis (C,D,H, I and J) showing the staining of sperm germ cells marker, Dazl (A,C), Primordial germ cell marker, Vasa (B,D), mature germ cell markers Actl7B (E,H), Hook1 (F,I) and Pgk2 (G,J). Double arrows on each of the wild-type sections (A,B,E,F,G) indicate the zone of differentiating spermatozoa absent in the Taz Neo tubules. Scale bar: 250 μ m

Article Snippet: Rabbit polyclonal anti-Hook1 (HPA018537) antibody, mouse monoclonal anti- β -tubulin isoform III (T5076), polyclonal anti-Actl7B (HPA021803) were purchased from Sigma-Aldrich, UK.

Techniques: Staining, Marker