anti ddx4 goat polyclonal  (R&D Systems)

Journal: eLife

Article Title: Amplification of a broad transcriptional program by a common factor triggers the meiotic cell cycle in mice

doi: 10.7554/eLife.43738

Figure Lengend Snippet: ( A ) Schematic for generation of the Stra8 FLAG allele. The FLAG sequence was inserted directly after the ATG in exon 2. ( B ) PCR genotyping for the Stra8 FLAG allele. ( C ) Western blot showing STRA8 (ab49405) and FLAG signal in protein lysates prepared from P10 wild-type and Stra8 FLAG/FLAG testes. α-tubulin was used as a loading control. The long STRA8 isoform, which is required for meiosis (see ), is tagged by FLAG. Stra8 FLAG/FLAG testes show reduced expression of the shorter STRA8 isoform, perhaps due to splicing changes caused by the FLAG insertion close to the start of exon 2. ( D ) Hematoxylin and periodic acid-Schiff (He/PAS)-stained sections from an 8 week Stra8 +/FLAG ovary, left, and a Stra8 FLAG/FLAG ovary, right. Scale bar = 100 μm. ( E ) Immunofluorescence (IF) staining for meiotic markers in E14.5 and E15.5 ovaries. Germ cells (marked by DDX4/VASA) in both Stra8 +/FLAG (left) and Stra8 FLAG/FLAG (right) ovaries express STRA8 and initiate meiosis; they show robust DMC1 and γ-H2AX staining. Scale bar = 10 μm. ( F ) He/PAS-stained testes from 8 week Stra8 +/FLAG and Stra8 FLAG/FLAG testes. Scale bar = 100 μm. ( G ) IF for meiotic markers in Stra8 +/FLAG and Stra8 FLAG/FLAG adult testes. In both genotypes, germ cells (marked by DDX4) initiate meiosis; they show STRA8, DMC1, and γ-H2AX staining. Scale bar = 20 μm. ( H ) IHC for STRA8 and FLAG, with hematoxylin counterstaining, of consecutive serial sections from an E14.5 Stra8 FLAG/FLAG fetal ovary. Scale bar = 200 μm. ( I ) FLAG IHC of the adult Stra8 FLAG/FLAG testis, with He/PAS counterstaining. Roman numerals indicate the seminiferous tubule stage. STRA8 FLAG is robustly expressed in preleptotene stage spermatocytes (yellow arrowheads) and in leptotene stage spermatocytes (green arrowheads). STRA8 FLAG is also expressed in spermatogonia (white arrowheads). Scale bar = 10 μm.

Article Snippet: Antibody , anti-DDX4/MVH (goat polyclonal) , R and D Systems , R and D Systems:AF2030; RRID: AB_2277369 , (IF 1:500).

Techniques: Sequencing, Western Blot, Control, Expressing, Staining, Immunofluorescence

Journal: eLife

Article Title: Amplification of a broad transcriptional program by a common factor triggers the meiotic cell cycle in mice

doi: 10.7554/eLife.43738

Figure Lengend Snippet: ( A ) Two Stra8 splice isoforms in wild-type mice. The Stra8 Δ 121 allele cannot express the longer protein isoform. The locations of peptides against which STRA8 antibodies were raised are marked. ( B ) STRA8 has a putative basic helix-loop-helix (bHLH) domain, a glutamic acid-rich region, and a putative high-mobility group (HMG) box domain (see Appendix 1). Shown are peptides recognized by antibodies, and regions required for nuclear localization and transactivation activity in vitro, as characterized by . ( C ) Western blots showing isoforms detected by STRA8 antibodies. Lysates were prepared from murine embryonic stem cells treated with DMSO (-) or RA (+) to induce STRA8 expression. ( D ) PCR genotyping of the Stra8 Δ 121 allele. ( E ) He/PAS-stained 8 week wild-type and Stra8 Δ121/Δ121 ovaries. Scale bar = 100 μm. ( F ) IF staining, in control ( Stra8 +/+ or Stra8 +/Δ121 ) and Stra8 Δ121/Δ121 embryonic ovaries, for the long STRA8 isoform (antibody ab49602), germ cell marker DDX4, and meiotic markers γ-H2AX, DMC1 and SCP3. Control germ cells express the long STRA8 isoform, are marked by γ-H2AX and DMC1 staining, and load the synaptonemal complex protein SCP3 onto condensed chromosomes. Stra8 Δ121/Δ121 germ cells, which do not express the long isoform, do not show these signs of meiotic initiation. Scale bars = 10 μm. ( G ) He/PAS staining of 3 month wild-type and Stra8 Δ121/Δ121 testes. Scale bar = 100 μm. ( H ) IF with antibodies for STRA8 (ab49602), γ-H2AX, DMC1, and DDX4 in adult testes (3 months). Scale bars = 20 μm. ( I ) STRA8 IHC, with hematoxylin counterstaining, of wild-type and Stra8 Δ121/Δ121 P12 testes. Staining of consecutive serial sections with the ab49602 and ab49405 antibodies indicates that Stra8 Δ121/Δ121 testes express only the shorter STRA8 isoform. Scale bar = 100 μm.

Article Snippet: Antibody , anti-DDX4/MVH (goat polyclonal) , R and D Systems , R and D Systems:AF2030; RRID: AB_2277369 , (IF 1:500).

Techniques: Activity Assay, In Vitro, Western Blot, Expressing, Staining, Control, Marker

Journal: eLife

Article Title: Amplification of a broad transcriptional program by a common factor triggers the meiotic cell cycle in mice

doi: 10.7554/eLife.43738

Figure Lengend Snippet: ( A ) IF staining of P12 testes using an antibody for the cytoplasmic germ cell marker DDX4 (VASA) and the STRA8 ab49405 antibody, which detects both STRA8 isoforms. Shown are images from a wild-type testis, which expresses both isoforms, and from a Stra8 Δ121/Δ121 testis, which expresses only the short isoform that is missing the first 111 amino acids. Scale bar = 20 μm. ( B ) Model representing the region of the longer STRA8 protein (393 aa) that is required for STRA8’s nuclear localization.

Article Snippet: Antibody , anti-DDX4/MVH (goat polyclonal) , R and D Systems , R and D Systems:AF2030; RRID: AB_2277369 , (IF 1:500).

Techniques: Staining, Marker

Journal: eLife

Article Title: Amplification of a broad transcriptional program by a common factor triggers the meiotic cell cycle in mice

doi: 10.7554/eLife.43738

Figure Lengend Snippet:

Article Snippet: Antibody , anti-DDX4/MVH (goat polyclonal) , R and D Systems , R and D Systems:AF2030; RRID: AB_2277369 , (IF 1:500).

Techniques: Knock-In, CRISPR, Generated, Recombinant, Plasmid Preparation, Sequencing, Software, Polymer, Sample Prep, Blocking Assay, Fluorescence, Protease Inhibitor, Immunoprecipitation, Western Blot, Lysis