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. 2019 May 31;9:8120. doi: 10.1038/s41598-019-44542-3

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© The Author(s) 2019

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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Both PfpTKL RVxF1 and RVxF2 motifs bind PfPP1c in vitro but only RVxF2 exerts a functional role in Xenopus laevis oocytes. (a) RVxF1 and RVxF2 both directly bind PfPP1c in vitro. PfPP1c-GST, PfeIf2γ-GST or GST (the last two = negative control) beads were incubated with His₆-pTKL_RVxF1 (RVxF1) or pTKL_KD_WT (KD_RVxF2). Following washes, beads were eluted in Laemmli buffer. Proteins were separated by SDS-PAGE and His₆-tagged proteins bound to beads were identified by western blot (WB, right panel – both blot first lanes show the specific and direct interaction of RVxF1 and KD_RVxF2 with PfPP1c-GST). All inputs were analyzed by western blot as well (left panel). Uncropped blots are available in the Supplementary Information. (b) Only RVxF2 binds XePP1c in vivo. RVxF1, KD_RVxF2 and KD_KRIMAS (containing RVxF2 inactivated by mutation) were micro-injected into X. laevis oocytes. To check for the presence of RVxF-containing proteins, oocytes were lysed and His₆-tagged proteins were separated by SDS-PAGE and detected by western blot (left panel). Oocytes micro-injected with RVxF1, KD_RVxF2 or KD_KRIMAS and incubated with progesterone were lysed 15 h after micro-injection and anti-XePP1c immunoprecipitation assays (IP) were performed. After washes, proteins bound to beads were detected by western blot (right panel). (c,d) RVxF2 inhibits progesterone-induced germinal vesicle breakdown (GVBD) in X. laevis oocytes. (c) Chart showing GVBD percentage over time in progesterone-incubated X. laevis oocytes micro-injected either with proteins or with control buffers (mean ± SD; three biological replicates and two technical replicates with 10 oocytes per data point). Oocyte images (external view and hemisections) are available for each sample in the Supplementary Information. ***, p < 0.001. (d) GVBD of oocytes micro-injected with RVxF-containing proteins was assayed by analyzing XeCDC2 dephosphorylation and XeH3 phosphorylation by western blot. Only KD_RVxF2 (third lane) was able to block progesterone-induced XeH3 phosphorylation. Lower panel, western blot analysis of total XeCDC2 and XeH3. Uncropped blots are available in the Supplementary Information.