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. 2017 Sep 18;7:11765. doi: 10.1038/s41598-017-12292-9

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© The Author(s) 2017

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

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α-SNAP expression in mouse ovarian tissue and granulosa cells (GCs) of wild type (WT) and α-SNAP mutant (hyh) females. (A) Western blot analysis of α-SNAP in postpubertal (postnatal day 60; P60) WT female mouse tissue extracts. GAPDH levels serve as loading control. Cropped blots (dotted lines) are displayed. Full-length blots including other tissue extracts are included in the Supplementary Figures file online (see Supplementary Figure S1). (B) Western blot analysis of α-SNAP in prepubertal (P7 and P14), peripubertal (P30), and postpubertal (P60 and P120) ovary extracts. β-tubulin levels serve as loading control. (C) Western blot analysis of α-SNAP in purified GCs and ovary remnants depleted of GCs (Re) at P30 and P60. N-cadherin (Ncad) was used as a marker of GC enrichment (see Supplementary Information) and Histone H3 was used as loading control. (A–C) Bars represent mean ± SEM of densitometric analyses (n = 3 or 4 independent experiments). (D–F) α-SNAP immunolabeling in P60 WT (E) and mutant hyh (F) ovarian follicles. Omission of the primary antibody was used as a negative control (D). Note the expression of α-SNAP in GCs. The expression of α-SNAP in the oocyte (O) cannot be established because of a non-specific fluorescent signal detected in the oocyte-zona pellucida region. (D) Scale bars, 10 μm. (D’–F’) Magnifications of GC regions in ovarian follicles. Images were pseudocolored using the lookup table shown at the right of the figures to highlight the differences in fluorescence intensity between WT (E’) and mutant hyh (F’) samples. Scale bars, 10 μm. (G) Quantification of α-SNAP immunofluorescence intensity in WT (black bar) and hyh (red bar) GCs. Bars represent mean ± SEM of 4 independent experiments. (H) Western blot images and densitometric analysis of α-SNAP in WT and hyh ovaries at P30 and P60. GAPDH was used as loading control. Note the hypomorphism of α-SNAP in hyh samples at both developmental stages. Bars represent mean ± SEM of densitometric analyses (n = 3 independent experiments). *p < 0.05; **p < 0.01; ***p < 0.001 (ANOVA with Tukey’s post hoc test or Student’s t-test).