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. 2021 Jul 9;12:4220. doi: 10.1038/s41467-021-24469-y

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© The Author(s) 2021

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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Fig. 1 — a, b Western blot analysis and densitometric quantification of Prok2 expression by ImageJ in brain tissue of control (n = 4 samples) and TBI (n = 9 samples) patients. Data presented as mean ± SD. c Immunofluorescence assessment of Prok2 expression (green) in brain tissue from control and TBI patients. Scale bar is 50 μm. DAPI is used to label nucleus. d Schematic representation of the contusional region (red) and the peri-contusional area (blue) after CCI. Tissues from the peri-contusional area (blue) are collected for western blot and qRT-PCR analysis. e, f Western blot analysis and densitometric quantification of Prok2 expression by ImageJ in control, sham and CCI mouse brain tissue. GAPDH is used as a control. Data presented as mean±SD (n = 3 mice). g Dual immunofluorescence staining shows that Prok2 expression is most prominent in neurons (NeuN-labeled), whereas low levels of Prok2-staining are found in astrocytes (GFAP-labeled) and microglia (iba-1-labeled). Scale bar is 15 μm. Quantification of Prok2 fluorescence intensity by ImageJ is shown in the right panel. Data presented as mean±SD (n = 3). h Representative photomicrographs of NeuN (red) and Map2 (green) expressing primary cortical neurons utilized in the studies. Scale bar is 50 μm for light microscopy image and 20 μm for fluorescence image. i Stretch-induced neuronal injury manifests as the appearance of thin and disrupted neurites and loss of the cytoplasm. Immunofluorescence labeling of tau protein (red) is used to monitor the effects of mechanical stretch on neurites; Hoechst is used to stain cell nuclei. Scale bar is 15 and 8 μm, respectively. j, k Western blot analysis and quantification of Prok2 and cleaved caspase-3 expression by ImageJ in control and stretch groups. GAPDH is used as loading control. Data presented as mean ± SD (n = 3 experiments). l, m Detection and quantification of Prok2 mRNA in primary cortical neurons exposed to Erastin for 24 h at different concentrations. β-actin is used as control. Data presented as mean ± SD (n = 3 experiments). n Erastin exposure increases Prok2 protein expression. GAPDH is used as control in western blot assays. For all panels, n indicates biologically independent repeats. P value was determined by a two-tailed unpaired Student’s t test for comparations between two groups. Source data are provided as a Source Data file.