Fig 1: Immunostaining PFA-fixed cryosections from neonatal and adult testes. (A) IIF was performed on cryosections from P6 testes, and specific primary antibodies used for each experiment are identified on each panel, with the colour of the text corresponding to the fluorescent secondary antibody employed. F-actin is labeled in all images using fluorescently-conjugated phalloidin. Antibodies used were: FOXO1 (Cell Signaling Technology, #2880), GFRA1 (R&D Systems, AF560), ZBTB16 (Santa Cruz Biotechnology, sc-22839), GATA4 (Santa Cruz Biotechnology, sc-1237), UCHL1 (Cell Signaling Technology, #D3T2E), CDH1 (Cell Signaling Technology, #3195), TRA98 (Abcam, ab82527), DDX4 (Abcam, ab13840), SOHLH1 (Pangas et al., 2006), STRA8 (Abcam, ab49602), SOHLH2 (Ballow et al., 2006), KIT (Santa Cruz Biotechnology, sc-1494), RHOX13 (Geyer and Eddy, 2008), phospho-RPS6 (Cell Signaling Technology, #5364). (B) IIF was performed on cryosections from P > 60 testes, and specific antibodies are indicated on each panel. Scale bars = 60 µm.
Fig 2: L1 ORF1p binding to endogenous mRNAs affects neither their stability nor their translation efficiency.(A) RNA-seq coverage of RNAs detected in anti-L1 ORF1p co-immunoprecipitation samples (IP and TOTAL; shown in triplicate). Each of the segments on the x axis (labeled 1 to 10) corresponds to 10% of transcript lengths; the cumulative number of reads across each segment is plotted (y axis, read count x106). (B) Immunofluorescence staining of L1 ORF1p (magenta) in P16 Mael+/- and Mael-/- testes. Scale bars: 100 µm. (C) Analysis of the poly(A) tail of Sycp1 and Setx mRNAs in P16 testes of Mael+/- and Mael-/- littermates. A schematic of the assay is shown on the left; numbers mark the assay steps: 1) poly(U) tailing; 2) cDNA synthesis using an RT adapter; and 3) PCR spanning the poly(A) tail. Representative gels are shown on the right; the assay was performed in biological triplicate. (D) Scatterplot showing the relationship between mRNA level changes (x axis, RNA-seq log2FoldChange) and ribo-footprint changes (y axis, Ribo-seq log2FoldChange) in Mael-/- over Mael+/- testes. A subset of 1313 mRNAs found associated with ORF1p previously in this study are labeled in red. ?TE n.s.: difference in Translation Efficiency between Mael-/- and Mael+/- non significant. (E) Western blot analysis of phosphorylated eIF2a levels (P-eIF2a) in P16 testes of Mael+/- and Mael-/- littermates. Blot #1 and #2 were run and processed in parallel. Blot #1 was first probed with an anti-P-eIF2a antibody, then stripped and re-probed with an anti-eIF2a antibody (pan); blot #2 was probed sequentially with the monoclonal anti-ORF1p (Abcam) and an anti-DDX4/MVH antibody.
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