probe  (ATCC)

Journal: Life Science Alliance

Article Title: Genetic mutation of Cep76 results in male infertility due to abnormal sperm tail composition

doi: 10.26508/lsa.202302452

Figure Lengend Snippet: (A) Proteins with significantly altered content in sperm from Cep76 mutants. (B) Proteins with significantly altered content in sperm from Cep76 mutants, after normalisation to alpha-tubulin content.

Article Snippet: To define the localisation of a subset of differentially expressed proteins, fixed sperm were permeabilised in 0.2% Triton X-100/PBS, blocked in CAS-Block, incubated overnight in primary antibodies (0.5 μg/ml DNAH2 [64309; Invitrogen], 2.5 μg/ml SEPT4 [166788; Abcam]) at 4°C, stained with relevant fluorescent secondary antibodies (Thermo Fisher Scientific) for 1 h, then counterstained with DAPI.

Techniques: Protease Inhibitor, Ubiquitin Proteomics, Clinical Proteomics, Membrane

Journal: Life Science Alliance

Article Title: Genetic mutation of Cep76 results in male infertility due to abnormal sperm tail composition

doi: 10.26508/lsa.202302452

Figure Lengend Snippet: (A) Wild-type versus Cep76 mutant data for (A) DNAH2 localisation in cauda epididymal sperm and (B) AKAP4 localisation in cauda epididymal sperm. Scale bars = 20 μm. Arrows point to the accumulation of DNAH2 or AKAP4 in the neck region of sperm. (C, D) Number of sperm with this neck localisation was quantified, as shown in (C, D), for DNAH2 and AKAP4, respectively. (E, F) Average tail pixel intensity (per area) of DNAH2 and AKAP4 was quantified and is shown in (E, F). *** P < 0.001, ** P < 0.01, * P < 0.05. All n = 3–5.

Article Snippet: To define the localisation of a subset of differentially expressed proteins, fixed sperm were permeabilised in 0.2% Triton X-100/PBS, blocked in CAS-Block, incubated overnight in primary antibodies (0.5 μg/ml DNAH2 [64309; Invitrogen], 2.5 μg/ml SEPT4 [166788; Abcam]) at 4°C, stained with relevant fluorescent secondary antibodies (Thermo Fisher Scientific) for 1 h, then counterstained with DAPI.

Techniques: Mutagenesis

Journal: eLife

Article Title: LRRC23 truncation impairs radial spoke 3 head assembly and sperm motility underlying male infertility

doi: 10.7554/eLife.90095

Figure Lengend Snippet: ( A ) Distribution of the axonemal components in Lrrc23 Δ/Δ sperm. Immunostained radial spoke (RSPH3B and RSPH9) and dynein arm (DNAH1, DNAH2, and DNAH9) in Lrrc23 +/Δ and Lrrc23 Δ/Δ sperm are shown by confocal images. Hoechst is used for counterstaining the sperm head. Experiment was performed with three biological replications. ( B, C ) Unaffected protein levels of the representative components of fibrous sheath, outer dense fiber, and axoneme in Lrrc23 Δ/Δ sperm. ( B ) Immunoblotting of the flagellar components in cauda sperm. Acetylated tubulin (AcTub) is a loading control. ( C ) Fold-changes of the flagellar components levels in Lrrc23 + /Δ (filled) and Lrrc23 Δ/Δ (sash) sperm. Relative protein levels were quantified by measuring the band intensity and normalized by the AcTub intensity. The average level of each protein in Lrrc23 +/Δ sperm is set to onefold. Circles represents fold changes of each sperm protein from individual males. Data represented as mean ± SEM. Statistical analysis was performed by Student’s t-test. N=3. ( D ) Transmission electron microscopy (TEM) images of epididymal sperm from Lrrc23 +/Δ and Lrrc23 Δ/Δ males. Shown are representative cross section TEM images of Lrrc23 +/Δ ( left ) and Lrrc23 Δ/Δ ( right ) sperm. No obvious structural defects at the midpiece (MP) and principal piece (PP) were observed from and Lrrc23 Δ/Δ sperm. M, mitochondria; FS, fibrous sheath; ODF, outer dense fiber; AX, axoneme. WT and/or Lrrc23 +/Δ sperm were used for positive control ( A, B, C and D ). Figure 4—figure supplement 1—source data 1. Uncropped blot images for .

Article Snippet: Rabbit polyclonal anti-LRRC23 (α–118, PA5-63449; α–208, PA5-58095), DNAH1 (PA5-57826), DNAH2 (PA5-64309), and DNAH9 (PA5-45744) were from Invitrogen.

Techniques: Western Blot, Control, Transmission Assay, Electron Microscopy, Positive Control