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OriGene ythdf2 plenti c mgfp p2a puro

Mn treatment decreases <t>YTHDF2</t> in cell culture (A) Primary mouse astrocytes were exposed to 100 μM for 1–24 h and YTHDF2 levels were determined by Western blot. The bottom panel shows the results of densitometric analysis of YTHDF2 bands normalized by β-actin (n = 4–5). YTHDF2 decreases time-dependently beginning after 3 h in primary mouse astrocytes. (B) ICC representation at 40x depicting YTHDF2 decreases in Mn-exposed primary mouse astrocytes at 24 h (100 μm scale). (C) 24-h treatment of human U251 astrocytes with different heavy metals, all at 100 μM ( n = 3). (D) Mn decreases YTHDF2 mRNA at 24 h in U251 astrocytes ( n = 5). (E) Mn decreases global m6A levels at 24 h in U251 astrocytes, as measured by LC-MS/MS ( n = 6). Data are means ± SEM. Two-group comparisons performed using unpaired t-test. p -values < 0.05 considered significant evidence.

Ythdf2 Plenti C Mgfp P2a Puro, supplied by OriGene, used in various techniques. Bioz Stars score: 92/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/product/ythdf2/YTHDF2+(NM_001173128)+Human+Tagged+ORF+Clone/pmc11382029-68-0-3
Average 92 stars, based on 1 article reviews

ythdf2 plenti c mgfp p2a puro - by Bioz Stars, 2026-09

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1) Product Images from "Epitranscriptomic reader YTHDF2 regulates SEK1( MAP2K4 )-JNK-cJUN inflammatory signaling in astrocytes during neurotoxic stress"

Article Title: Epitranscriptomic reader YTHDF2 regulates SEK1( MAP2K4 )-JNK-cJUN inflammatory signaling in astrocytes during neurotoxic stress

Journal: iScience

doi: 10.1016/j.isci.2024.110619

Mn treatment decreases YTHDF2 in cell culture (A) Primary mouse astrocytes were exposed to 100 μM for 1–24 h and YTHDF2 levels were determined by Western blot. The bottom panel shows the results of densitometric analysis of YTHDF2 bands normalized by β-actin (n = 4–5). YTHDF2 decreases time-dependently beginning after 3 h in primary mouse astrocytes. (B) ICC representation at 40x depicting YTHDF2 decreases in Mn-exposed primary mouse astrocytes at 24 h (100 μm scale). (C) 24-h treatment of human U251 astrocytes with different heavy metals, all at 100 μM ( n = 3). (D) Mn decreases YTHDF2 mRNA at 24 h in U251 astrocytes ( n = 5). (E) Mn decreases global m6A levels at 24 h in U251 astrocytes, as measured by LC-MS/MS ( n = 6). Data are means ± SEM. Two-group comparisons performed using unpaired t-test. p -values < 0.05 considered significant evidence.

Figure Legend Snippet: Mn treatment decreases YTHDF2 in cell culture (A) Primary mouse astrocytes were exposed to 100 μM for 1–24 h and YTHDF2 levels were determined by Western blot. The bottom panel shows the results of densitometric analysis of YTHDF2 bands normalized by β-actin (n = 4–5). YTHDF2 decreases time-dependently beginning after 3 h in primary mouse astrocytes. (B) ICC representation at 40x depicting YTHDF2 decreases in Mn-exposed primary mouse astrocytes at 24 h (100 μm scale). (C) 24-h treatment of human U251 astrocytes with different heavy metals, all at 100 μM ( n = 3). (D) Mn decreases YTHDF2 mRNA at 24 h in U251 astrocytes ( n = 5). (E) Mn decreases global m6A levels at 24 h in U251 astrocytes, as measured by LC-MS/MS ( n = 6). Data are means ± SEM. Two-group comparisons performed using unpaired t-test. p -values < 0.05 considered significant evidence.

Techniques Used: Cell Culture, Western Blot, Liquid Chromatography with Mass Spectroscopy

YTHDF2 levels can affect pro-inflammatory chemokine/cytokine responses in Mn-exposed astrocytes (A) Validation of YTHDF2 knockdown using siRNA, both qPCR and immunoblotting (n = 5–6). (B) Validation of YTHDF2 overexpression, both qPCR and immunoblotting ( n = 3). (C) siYTHDF2 increased basal pro-inflammatory gene expression and exacerbated after Mn exposure ( n = 3). (D) Overexpression of YTHDF2 suppressed basal pro-inflammatory gene expression and prevented upregulation after Mn exposure ( n = 4). (E and F) Multiplex ELISA analysis of the treatment media of siYTHDF2 and YTHDF2 overexpression experiments for cytokines/chemokines (n = 4–6). IL-8 was most consistently affected by YTHDF2 levels, showing exacerbated release in siYTHDF2 experiments, while its release was prevented in YTHDF2 overexpression experiments. MCP1 (MCAF), IFNγ, IL-6, and MIP1b showed similar trends but with less consistency overall. Data are means ± SEM. Two-group comparisons performed using unpaired t-test. p -values < 0.05 considered significant evidence. Two-way ANOVA with FDR Two-stage step-up method of Benjamini, Krieger, and Yekutieli for multi-group comparison. Q-values < 0.05 considered significant evidence.

Figure Legend Snippet: YTHDF2 levels can affect pro-inflammatory chemokine/cytokine responses in Mn-exposed astrocytes (A) Validation of YTHDF2 knockdown using siRNA, both qPCR and immunoblotting (n = 5–6). (B) Validation of YTHDF2 overexpression, both qPCR and immunoblotting ( n = 3). (C) siYTHDF2 increased basal pro-inflammatory gene expression and exacerbated after Mn exposure ( n = 3). (D) Overexpression of YTHDF2 suppressed basal pro-inflammatory gene expression and prevented upregulation after Mn exposure ( n = 4). (E and F) Multiplex ELISA analysis of the treatment media of siYTHDF2 and YTHDF2 overexpression experiments for cytokines/chemokines (n = 4–6). IL-8 was most consistently affected by YTHDF2 levels, showing exacerbated release in siYTHDF2 experiments, while its release was prevented in YTHDF2 overexpression experiments. MCP1 (MCAF), IFNγ, IL-6, and MIP1b showed similar trends but with less consistency overall. Data are means ± SEM. Two-group comparisons performed using unpaired t-test. p -values < 0.05 considered significant evidence. Two-way ANOVA with FDR Two-stage step-up method of Benjamini, Krieger, and Yekutieli for multi-group comparison. Q-values < 0.05 considered significant evidence.

Techniques Used: Biomarker Discovery, Knockdown, Western Blot, Over Expression, Gene Expression, Multiplex Assay, Enzyme-linked Immunosorbent Assay, Comparison

RNA- and RIP-sequencing of Mn-exposed U251 astrocytes reveals YTHDF2 targeting of MAP2K4 (SEK1) (A and B) (A) Z-scores of Mn/Ctrl GO for Transcriptional Factor Targets, indicating important roles for cJUN and HIF1α (B) RIP-sequencing graphical representation of statistically significant YTHDF2 targets in Ctrl that were affected by Mn exposure. YTHDF2 targets are identified as having ≥+1 log2(RIP/input) ratio. MAP2K4 was identified as a lost YTHDF2 target under Mn exposure, suggesting regulation of the SEK1( MAP2K4 )-JNK-cJUN pathway. (C) si YTHDF2 astrocytes present with longer MAP2K4 mRNA half-life ( n = 3). (D) ICC representation at 40x depicting si YTHDF2 astrocytes have increased SEK1 protein levels (100 μm scale). (E) YTHDF2 overexpressing astrocytes present with shorter MAP2K4 half-life under Mn exposure (n = 4–5). (F) ICC representation at 40x depicting YTHDF2-overexpressing astrocytes have decreased SEK1 protein levels (100 μm scale). (G and H) Immunoblotting and quantification revealing cJUN phosphorylation is increased in Mn-exposed astrocytes and sustained in siYTHDF2 Mn-exposed astrocytes. SEK1 protein levels are basally increased in siYTHDF2 astrocytes (n = 3–4). (I and J) Immunoblotting and quantification revealing cJUN phosphorylation is increased in Mn-exposed astrocytes and prevented in YTHDF2-overexpressing Mn-exposed astrocytes. SEK1 protein levels are basally decreased in YTHDF2-overexpressing astrocytes ( n = 4). Data are means ± SEM. Two-group comparisons performed using unpaired t-test, with 2-fold gene threshold and 1.96 Z - score threshold using Altanalyze. Adjusted p -values < 0.05 considered significant evidence. For mRNA half-life comparisons, One-phase decay non-linear regression analysis was performed. p -values < 0.05 considered significant evidence. two-way ANOVA with FDR Two-stage step-up method of Benjamini, Krieger, and Yekutieli for multi-group comparison. Q-values < 0.05 considered significant evidence.

Figure Legend Snippet: RNA- and RIP-sequencing of Mn-exposed U251 astrocytes reveals YTHDF2 targeting of MAP2K4 (SEK1) (A and B) (A) Z-scores of Mn/Ctrl GO for Transcriptional Factor Targets, indicating important roles for cJUN and HIF1α (B) RIP-sequencing graphical representation of statistically significant YTHDF2 targets in Ctrl that were affected by Mn exposure. YTHDF2 targets are identified as having ≥+1 log2(RIP/input) ratio. MAP2K4 was identified as a lost YTHDF2 target under Mn exposure, suggesting regulation of the SEK1( MAP2K4 )-JNK-cJUN pathway. (C) si YTHDF2 astrocytes present with longer MAP2K4 mRNA half-life ( n = 3). (D) ICC representation at 40x depicting si YTHDF2 astrocytes have increased SEK1 protein levels (100 μm scale). (E) YTHDF2 overexpressing astrocytes present with shorter MAP2K4 half-life under Mn exposure (n = 4–5). (F) ICC representation at 40x depicting YTHDF2-overexpressing astrocytes have decreased SEK1 protein levels (100 μm scale). (G and H) Immunoblotting and quantification revealing cJUN phosphorylation is increased in Mn-exposed astrocytes and sustained in siYTHDF2 Mn-exposed astrocytes. SEK1 protein levels are basally increased in siYTHDF2 astrocytes (n = 3–4). (I and J) Immunoblotting and quantification revealing cJUN phosphorylation is increased in Mn-exposed astrocytes and prevented in YTHDF2-overexpressing Mn-exposed astrocytes. SEK1 protein levels are basally decreased in YTHDF2-overexpressing astrocytes ( n = 4). Data are means ± SEM. Two-group comparisons performed using unpaired t-test, with 2-fold gene threshold and 1.96 Z - score threshold using Altanalyze. Adjusted p -values < 0.05 considered significant evidence. For mRNA half-life comparisons, One-phase decay non-linear regression analysis was performed. p -values < 0.05 considered significant evidence. two-way ANOVA with FDR Two-stage step-up method of Benjamini, Krieger, and Yekutieli for multi-group comparison. Q-values < 0.05 considered significant evidence.

Techniques Used: Sequencing, Western Blot, Phospho-proteomics, Comparison

YTHDF2 is decreased in Mn-gavaged mice, and a conditional knockout of YTHDF2 in the astrocytes of mice leads to increased astrocyte reactivity in substantia nigra pars compacta/reticulata and globus pallidus (A) IHC representation at 40x depicting YTHDF2 decreases and colocalization in GFAP+ cells in the globus pallidus of Mn-gavaged mice (100 μm scale). (B) Immunoblotting of the substantia nigra showing decreases of YTHDF2 in mice gavaged with Mn ( n = 9). (C) Schematic of Y2cKO mice generation. Tamoxifen (tiY2cKO) induces Cre recombination of YTHDF2 exon 4 to produce a band at 748 bp in astrocytes isolated from striatal/hippocampal brain tissue using biotinylated-EAAT1/GLAST-1 antibody. (D) Globus pallidus validation of YTHDF2 protein reduction and increased GFAP immunoreactivity, and also increased m6A staining upon YTHDF2 deletion. YTHDF2 was quantified by parent-child extraction using GFAP as the parent (n = 3–4) (100 μm scale). (E) Globus pallidus representative images of increased C3d immunoreactivity in GFAP-positive cells in the tiY2cKO mice (100 μm scale). (F) Immunoblotting and quantification of substantia nigra- showing tiY2cKO mice with increased GFAP immunoreactivity similar to Mn-gavaged mice, indicating specific loss of YTHDF2 in astrocytes increases their reactivity (n = 4–6). (G) Substantia nigra representative images of increased C3d immunoreactivity in GFAP-positive cells in the tiY2cKO mice (100 μm scale). Data are means ± SEM. Student’s t test or two-way ANOVA with FDR Two-stage step-up method of Benjamini, Krieger, and Yekutieli for multi-group comparison. Q-values < 0.05 considered significant evidence, with q-values between 0.05 and 0.01 considered as weaker evidence taking into consideration variations in data and trends.

Figure Legend Snippet: YTHDF2 is decreased in Mn-gavaged mice, and a conditional knockout of YTHDF2 in the astrocytes of mice leads to increased astrocyte reactivity in substantia nigra pars compacta/reticulata and globus pallidus (A) IHC representation at 40x depicting YTHDF2 decreases and colocalization in GFAP+ cells in the globus pallidus of Mn-gavaged mice (100 μm scale). (B) Immunoblotting of the substantia nigra showing decreases of YTHDF2 in mice gavaged with Mn ( n = 9). (C) Schematic of Y2cKO mice generation. Tamoxifen (tiY2cKO) induces Cre recombination of YTHDF2 exon 4 to produce a band at 748 bp in astrocytes isolated from striatal/hippocampal brain tissue using biotinylated-EAAT1/GLAST-1 antibody. (D) Globus pallidus validation of YTHDF2 protein reduction and increased GFAP immunoreactivity, and also increased m6A staining upon YTHDF2 deletion. YTHDF2 was quantified by parent-child extraction using GFAP as the parent (n = 3–4) (100 μm scale). (E) Globus pallidus representative images of increased C3d immunoreactivity in GFAP-positive cells in the tiY2cKO mice (100 μm scale). (F) Immunoblotting and quantification of substantia nigra- showing tiY2cKO mice with increased GFAP immunoreactivity similar to Mn-gavaged mice, indicating specific loss of YTHDF2 in astrocytes increases their reactivity (n = 4–6). (G) Substantia nigra representative images of increased C3d immunoreactivity in GFAP-positive cells in the tiY2cKO mice (100 μm scale). Data are means ± SEM. Student’s t test or two-way ANOVA with FDR Two-stage step-up method of Benjamini, Krieger, and Yekutieli for multi-group comparison. Q-values < 0.05 considered significant evidence, with q-values between 0.05 and 0.01 considered as weaker evidence taking into consideration variations in data and trends.

Techniques Used: Knock-Out, Western Blot, Isolation, Biomarker Discovery, Staining, Extraction, Comparison

Integrated working hypothesis of YTHDF2’s role on the SEK1( MAP2K4 )-JNK-cJUN pathway Upon Mn exposure, ALKBH5 levels increase while YTHDF2 decreases, leading to increased half-life of MAP2K4 mRNA. Increased abundance of MAP2K4 leads to more SEK1 protein, allowing for the sustained activation (increased phosphorylation) of the downstream pathway targets JNK and cJUN by Mn, which promotes the pro-inflammatory response in astrocytes. When YTHDF2 is overexpressed, MAP2K4 mRNA is degraded. This leads to lower SEK1 protein levels, reducing the pathway activation of JNK and cJUN (decreased phosphorylation) by Mn, resulting in an anti-inflammatory response.

Figure Legend Snippet: Integrated working hypothesis of YTHDF2’s role on the SEK1( MAP2K4 )-JNK-cJUN pathway Upon Mn exposure, ALKBH5 levels increase while YTHDF2 decreases, leading to increased half-life of MAP2K4 mRNA. Increased abundance of MAP2K4 leads to more SEK1 protein, allowing for the sustained activation (increased phosphorylation) of the downstream pathway targets JNK and cJUN by Mn, which promotes the pro-inflammatory response in astrocytes. When YTHDF2 is overexpressed, MAP2K4 mRNA is degraded. This leads to lower SEK1 protein levels, reducing the pathway activation of JNK and cJUN (decreased phosphorylation) by Mn, resulting in an anti-inflammatory response.

Techniques Used: Activation Assay, Phospho-proteomics

Figure Legend Snippet:

Techniques Used: Recombinant, Extraction, Selection, Negative Control, Software

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