egr3  (Cell Signaling Technology Inc)


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    Cell Signaling Technology Inc egr3
    (A)Immunoblots for Egr1, Egr2 and <t>Egr3</t> before or at the indicated time points after the stimulation with plate-bound anti-CD3ε and anti-CD28 antibodies in DP cells from the indicated genotypes. Actin was used as a loading control. Data are representative of three independent experiments. (B) Immunoblots for Egr1, Egr2 and Egr3 in DP cells from the indicated genotypes before or at the indicated time points after the stimulation with plate-bound anti-CD3ε and anti-CD28 antibodies under the condition with U0126 in DMSO or with DMSO alone. Actin was used as a loading control. Data are representative of three independent experiments. (C, D) Quantitative RT-PCR analysis of Egr1 , Egr2 and Egr3 expression before (C) or at the indicated time points after the stimulation with anti-CD3ε and anti-CD28 antibodies (D) in DP cells from Zfat f/f and Zfat f/f - LckCre mice. The relative expression for each gene was normalized by the expression of Actb . The results are presented as the value relative to the unstimulated DP cells from Zfat f/f mice. The data are the mean ± s.d.; n.s., not significant.
    Egr3, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    1) Product Images from "Zfat -Deficiency Results in a Loss of CD3ζ Phosphorylation with Dysregulation of ERK and Egr Activities Leading to Impaired Positive Selection"

    Article Title: Zfat -Deficiency Results in a Loss of CD3ζ Phosphorylation with Dysregulation of ERK and Egr Activities Leading to Impaired Positive Selection

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0076254

    (A)Immunoblots for Egr1, Egr2 and Egr3 before or at the indicated time points after the stimulation with plate-bound anti-CD3ε and anti-CD28 antibodies in DP cells from the indicated genotypes. Actin was used as a loading control. Data are representative of three independent experiments. (B) Immunoblots for Egr1, Egr2 and Egr3 in DP cells from the indicated genotypes before or at the indicated time points after the stimulation with plate-bound anti-CD3ε and anti-CD28 antibodies under the condition with U0126 in DMSO or with DMSO alone. Actin was used as a loading control. Data are representative of three independent experiments. (C, D) Quantitative RT-PCR analysis of Egr1 , Egr2 and Egr3 expression before (C) or at the indicated time points after the stimulation with anti-CD3ε and anti-CD28 antibodies (D) in DP cells from Zfat f/f and Zfat f/f - LckCre mice. The relative expression for each gene was normalized by the expression of Actb . The results are presented as the value relative to the unstimulated DP cells from Zfat f/f mice. The data are the mean ± s.d.; n.s., not significant.
    Figure Legend Snippet: (A)Immunoblots for Egr1, Egr2 and Egr3 before or at the indicated time points after the stimulation with plate-bound anti-CD3ε and anti-CD28 antibodies in DP cells from the indicated genotypes. Actin was used as a loading control. Data are representative of three independent experiments. (B) Immunoblots for Egr1, Egr2 and Egr3 in DP cells from the indicated genotypes before or at the indicated time points after the stimulation with plate-bound anti-CD3ε and anti-CD28 antibodies under the condition with U0126 in DMSO or with DMSO alone. Actin was used as a loading control. Data are representative of three independent experiments. (C, D) Quantitative RT-PCR analysis of Egr1 , Egr2 and Egr3 expression before (C) or at the indicated time points after the stimulation with anti-CD3ε and anti-CD28 antibodies (D) in DP cells from Zfat f/f and Zfat f/f - LckCre mice. The relative expression for each gene was normalized by the expression of Actb . The results are presented as the value relative to the unstimulated DP cells from Zfat f/f mice. The data are the mean ± s.d.; n.s., not significant.

    Techniques Used: Western Blot, Quantitative RT-PCR, Expressing

    egr3  (Cell Signaling Technology Inc)


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    Cell Signaling Technology Inc egr3
    (A)Immunoblots for Egr1, Egr2 and <t>Egr3</t> before or at the indicated time points after the stimulation with plate-bound anti-CD3ε and anti-CD28 antibodies in DP cells from the indicated genotypes. Actin was used as a loading control. Data are representative of three independent experiments. (B) Immunoblots for Egr1, Egr2 and Egr3 in DP cells from the indicated genotypes before or at the indicated time points after the stimulation with plate-bound anti-CD3ε and anti-CD28 antibodies under the condition with U0126 in DMSO or with DMSO alone. Actin was used as a loading control. Data are representative of three independent experiments. (C, D) Quantitative RT-PCR analysis of Egr1 , Egr2 and Egr3 expression before (C) or at the indicated time points after the stimulation with anti-CD3ε and anti-CD28 antibodies (D) in DP cells from Zfat f/f and Zfat f/f - LckCre mice. The relative expression for each gene was normalized by the expression of Actb . The results are presented as the value relative to the unstimulated DP cells from Zfat f/f mice. The data are the mean ± s.d.; n.s., not significant.
    Egr3, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    1) Product Images from "Zfat -Deficiency Results in a Loss of CD3ζ Phosphorylation with Dysregulation of ERK and Egr Activities Leading to Impaired Positive Selection"

    Article Title: Zfat -Deficiency Results in a Loss of CD3ζ Phosphorylation with Dysregulation of ERK and Egr Activities Leading to Impaired Positive Selection

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0076254

    (A)Immunoblots for Egr1, Egr2 and Egr3 before or at the indicated time points after the stimulation with plate-bound anti-CD3ε and anti-CD28 antibodies in DP cells from the indicated genotypes. Actin was used as a loading control. Data are representative of three independent experiments. (B) Immunoblots for Egr1, Egr2 and Egr3 in DP cells from the indicated genotypes before or at the indicated time points after the stimulation with plate-bound anti-CD3ε and anti-CD28 antibodies under the condition with U0126 in DMSO or with DMSO alone. Actin was used as a loading control. Data are representative of three independent experiments. (C, D) Quantitative RT-PCR analysis of Egr1 , Egr2 and Egr3 expression before (C) or at the indicated time points after the stimulation with anti-CD3ε and anti-CD28 antibodies (D) in DP cells from Zfat f/f and Zfat f/f - LckCre mice. The relative expression for each gene was normalized by the expression of Actb . The results are presented as the value relative to the unstimulated DP cells from Zfat f/f mice. The data are the mean ± s.d.; n.s., not significant.
    Figure Legend Snippet: (A)Immunoblots for Egr1, Egr2 and Egr3 before or at the indicated time points after the stimulation with plate-bound anti-CD3ε and anti-CD28 antibodies in DP cells from the indicated genotypes. Actin was used as a loading control. Data are representative of three independent experiments. (B) Immunoblots for Egr1, Egr2 and Egr3 in DP cells from the indicated genotypes before or at the indicated time points after the stimulation with plate-bound anti-CD3ε and anti-CD28 antibodies under the condition with U0126 in DMSO or with DMSO alone. Actin was used as a loading control. Data are representative of three independent experiments. (C, D) Quantitative RT-PCR analysis of Egr1 , Egr2 and Egr3 expression before (C) or at the indicated time points after the stimulation with anti-CD3ε and anti-CD28 antibodies (D) in DP cells from Zfat f/f and Zfat f/f - LckCre mice. The relative expression for each gene was normalized by the expression of Actb . The results are presented as the value relative to the unstimulated DP cells from Zfat f/f mice. The data are the mean ± s.d.; n.s., not significant.

    Techniques Used: Western Blot, Quantitative RT-PCR, Expressing

    egr3  (Cell Signaling Technology Inc)


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    Cell Signaling Technology Inc egr3
    Regulators of cellular stress and growth response are differently expressed in heat-shocked HSF1+ and HSF1− cells. ( A ) Expression of HSPA1 , ATF3 , JUNB , FOSB , EGR1 , and <t>EGR3</t> analyzed by RT-qPCR in MCF7, HAP1, and RKO cells exposed to elevated temperature (HS: 43 °C/1 h + recovery 37 °C/2 h, 4 h, or 6 h) in relation (fold change) to untreated control (Ctr) in HSF1+ cells. In the case of MCF7, a different cell model was used than for RNA-seq. Statistically significant differences between HSF1+ and HSF1− in each time point: ** p < 0.001, * p < 0.05. ( B ) Western blot analyses of HSF1 (the asterisk shows non-specific bands) in untreated HSF1+ and HSF1− MCF7, HAP1, and RKO cells and HSPA1, ATF3, and EGR3 up to 24 h of recovery from one-hour heat shock. ACTB or HSPA8 were used as loading controls. Results of densitometric analyses (n = 2–4) are shown below blots (note that highly variable EGR3 expression resulted in large standard deviations). Statistically significant differences between HSF1+ and HSF1− in each time point: ** p < 0.001, * p < 0.05.
    Egr3, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    1) Product Images from "HSF1 Can Prevent Inflammation following Heat Shock by Inhibiting the Excessive Activation of the ATF3 and JUN & FOS Genes"

    Article Title: HSF1 Can Prevent Inflammation following Heat Shock by Inhibiting the Excessive Activation of the ATF3 and JUN & FOS Genes

    Journal: Cells

    doi: 10.3390/cells11162510

    Regulators of cellular stress and growth response are differently expressed in heat-shocked HSF1+ and HSF1− cells. ( A ) Expression of HSPA1 , ATF3 , JUNB , FOSB , EGR1 , and EGR3 analyzed by RT-qPCR in MCF7, HAP1, and RKO cells exposed to elevated temperature (HS: 43 °C/1 h + recovery 37 °C/2 h, 4 h, or 6 h) in relation (fold change) to untreated control (Ctr) in HSF1+ cells. In the case of MCF7, a different cell model was used than for RNA-seq. Statistically significant differences between HSF1+ and HSF1− in each time point: ** p < 0.001, * p < 0.05. ( B ) Western blot analyses of HSF1 (the asterisk shows non-specific bands) in untreated HSF1+ and HSF1− MCF7, HAP1, and RKO cells and HSPA1, ATF3, and EGR3 up to 24 h of recovery from one-hour heat shock. ACTB or HSPA8 were used as loading controls. Results of densitometric analyses (n = 2–4) are shown below blots (note that highly variable EGR3 expression resulted in large standard deviations). Statistically significant differences between HSF1+ and HSF1− in each time point: ** p < 0.001, * p < 0.05.
    Figure Legend Snippet: Regulators of cellular stress and growth response are differently expressed in heat-shocked HSF1+ and HSF1− cells. ( A ) Expression of HSPA1 , ATF3 , JUNB , FOSB , EGR1 , and EGR3 analyzed by RT-qPCR in MCF7, HAP1, and RKO cells exposed to elevated temperature (HS: 43 °C/1 h + recovery 37 °C/2 h, 4 h, or 6 h) in relation (fold change) to untreated control (Ctr) in HSF1+ cells. In the case of MCF7, a different cell model was used than for RNA-seq. Statistically significant differences between HSF1+ and HSF1− in each time point: ** p < 0.001, * p < 0.05. ( B ) Western blot analyses of HSF1 (the asterisk shows non-specific bands) in untreated HSF1+ and HSF1− MCF7, HAP1, and RKO cells and HSPA1, ATF3, and EGR3 up to 24 h of recovery from one-hour heat shock. ACTB or HSPA8 were used as loading controls. Results of densitometric analyses (n = 2–4) are shown below blots (note that highly variable EGR3 expression resulted in large standard deviations). Statistically significant differences between HSF1+ and HSF1− in each time point: ** p < 0.001, * p < 0.05.

    Techniques Used: Expressing, Quantitative RT-PCR, RNA Sequencing Assay, Western Blot

    egr3  (Cell Signaling Technology Inc)


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    Cell Signaling Technology Inc egr3

    Egr3, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    1) Product Images from "Heat shock factor 1 (HSF1) cooperates with estrogen receptor α (ERα) in the regulation of estrogen action in breast cancer cells"

    Article Title: Heat shock factor 1 (HSF1) cooperates with estrogen receptor α (ERα) in the regulation of estrogen action in breast cancer cells

    Journal: eLife

    doi: 10.7554/eLife.69843


    Figure Legend Snippet:

    Techniques Used: Transfection, Construct, Recombinant, Plasmid Preparation, shRNA, Expressing, Sequencing, In Situ, SYBR Green Assay, Software, Staining, CRISPR, Protease Inhibitor

    egr3 antibody  (Cell Signaling Technology Inc)


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    Cell Signaling Technology Inc egr3 antibody
    Patient characteristics.
    Egr3 Antibody, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    1) Product Images from "Expression and prognostic value of the transcription factors EGR1 and EGR3 in gliomas"

    Article Title: Expression and prognostic value of the transcription factors EGR1 and EGR3 in gliomas

    Journal: Scientific Reports

    doi: 10.1038/s41598-020-66236-x

    Patient characteristics.
    Figure Legend Snippet: Patient characteristics.

    Techniques Used: Methylation

    EGR1 and EGR3 protein expression in gliomas. (A) EGR1 and EGR3 expression levels displayed in representative grade II, III and IV gliomas. Images were acquired at 40X magnification with scale bars = 50 μm. EGR1 protein expression significantly increased with WHO grade and the highest expression levels were found in glioblastomas. EGR3 protein expression levels were independent of WHO grade. ( B) Expression of both EGR1 and EGR3 was seen in areas with characteristic histological traits of GBMs, including areas with necrosis, pseudopalisades and around microvascular proliferations. Images were acquired at 30X magnification with scale bars = 100 μm. N = Necrosis. P = pseudo-palisade. M = microvascular proliferation. ( C) Verhaak subtype stratification of EGR1 mRNA TCGA data showed that neural and proneural glioblastomas have slightly lower EGR1 mRNA levels compared to classical and mesenchymal tumors, while EGR3 was equally expressed in all subtypes. ( D) Stratification of EGR1 and EGR3 protein levels based on IDH1/2 mutational status in the patient cohort. No significant changes in EGR1 or EGR3 protein levels were observed. * = P < 0.05. ** = P < 0.01. *** = P < 0.001.
    Figure Legend Snippet: EGR1 and EGR3 protein expression in gliomas. (A) EGR1 and EGR3 expression levels displayed in representative grade II, III and IV gliomas. Images were acquired at 40X magnification with scale bars = 50 μm. EGR1 protein expression significantly increased with WHO grade and the highest expression levels were found in glioblastomas. EGR3 protein expression levels were independent of WHO grade. ( B) Expression of both EGR1 and EGR3 was seen in areas with characteristic histological traits of GBMs, including areas with necrosis, pseudopalisades and around microvascular proliferations. Images were acquired at 30X magnification with scale bars = 100 μm. N = Necrosis. P = pseudo-palisade. M = microvascular proliferation. ( C) Verhaak subtype stratification of EGR1 mRNA TCGA data showed that neural and proneural glioblastomas have slightly lower EGR1 mRNA levels compared to classical and mesenchymal tumors, while EGR3 was equally expressed in all subtypes. ( D) Stratification of EGR1 and EGR3 protein levels based on IDH1/2 mutational status in the patient cohort. No significant changes in EGR1 or EGR3 protein levels were observed. * = P < 0.05. ** = P < 0.01. *** = P < 0.001.

    Techniques Used: Expressing

    Expression of EGR1 and EGR3 in infiltrating tumour cells. (A–C) Representative HE, P53, and double-fluorescence images with P53/EGR1 on tissue sections from one of the tumours included in the cohort. 1 = central tumour area. 2 = intermediate tumour area. 3 = peripheral tumour area. Scale bar = 2.5 mm. DAPI-stained nuclei = blue, P53-positive nuclei = green, EGR1/3-positive nuclei = red, double-positive nuclei, i.e. positive for both P53 and EGR1/3 = orange. ( D,E) The EGR1 fraction and staining intensity in EGR1 + tumour cells was significantly reduced from central tumour to periphery. ( F) Ivy GAP data showed a trending but non-significant decline in EGR1 mRNA levels between central tumour and tumour periphery. ( G,H) The EGR3 fraction remained constant across the different tumour regions, while the mean staining intensity increased significantly in peripheral tumour cells compared to central tumour cells. I) Ivy Gap EGR3 mRNA data also showed an increase in EGR3 intensity in the intermediate and peripheral areas compared to central tumour. * = P < 0.05. ** = P < 0.01. *** = P < 0.001.
    Figure Legend Snippet: Expression of EGR1 and EGR3 in infiltrating tumour cells. (A–C) Representative HE, P53, and double-fluorescence images with P53/EGR1 on tissue sections from one of the tumours included in the cohort. 1 = central tumour area. 2 = intermediate tumour area. 3 = peripheral tumour area. Scale bar = 2.5 mm. DAPI-stained nuclei = blue, P53-positive nuclei = green, EGR1/3-positive nuclei = red, double-positive nuclei, i.e. positive for both P53 and EGR1/3 = orange. ( D,E) The EGR1 fraction and staining intensity in EGR1 + tumour cells was significantly reduced from central tumour to periphery. ( F) Ivy GAP data showed a trending but non-significant decline in EGR1 mRNA levels between central tumour and tumour periphery. ( G,H) The EGR3 fraction remained constant across the different tumour regions, while the mean staining intensity increased significantly in peripheral tumour cells compared to central tumour cells. I) Ivy Gap EGR3 mRNA data also showed an increase in EGR3 intensity in the intermediate and peripheral areas compared to central tumour. * = P < 0.05. ** = P < 0.01. *** = P < 0.001.

    Techniques Used: Expressing, Fluorescence, Staining

    Overall patient survival in different subgroups. Patient survival illustrated for different EGR1 and EGR3 subgroups in glioblastoma patients both measured by positive cell fractions found in the immunostainings and by mRNA expression data from TCGA. All groups were dichotomized at the median. ( A) GBM patients with a high EGR1 nuclear fraction showed a significantly better prognosis compared to patients with a low EGR1 fraction. ( B) EGR1 mRNA levels from TCGA did not show any difference in survival between the two groups. ( C) Patients with a high EGR3 nuclear fraction were borderline significant of having a poor prognosis compared to patients with a low EGR3 fraction. ( D) EGR3 mRNA levels from TCGA showed the same trend as EGR3 protein levels. ( E) MGMT-gene promoter methylated patients with a high EGR3 fraction lived significantly shorter than patients with a low EGR3 fraction. ( F) The same trend was found in EGR3 mRNA data from TCGA, although results were non-significant . G) When sub-stratifying patients from the EGR1 high group by EGR3 expression, the group with high EGR3 expression had significantly shorter survival compared to patients with low EGR3 expression. H) Sub-stratification of patients from the EGR1 low group by EGR3 expression did not result in survival differences between the two groups. HR = Hazard ratio.
    Figure Legend Snippet: Overall patient survival in different subgroups. Patient survival illustrated for different EGR1 and EGR3 subgroups in glioblastoma patients both measured by positive cell fractions found in the immunostainings and by mRNA expression data from TCGA. All groups were dichotomized at the median. ( A) GBM patients with a high EGR1 nuclear fraction showed a significantly better prognosis compared to patients with a low EGR1 fraction. ( B) EGR1 mRNA levels from TCGA did not show any difference in survival between the two groups. ( C) Patients with a high EGR3 nuclear fraction were borderline significant of having a poor prognosis compared to patients with a low EGR3 fraction. ( D) EGR3 mRNA levels from TCGA showed the same trend as EGR3 protein levels. ( E) MGMT-gene promoter methylated patients with a high EGR3 fraction lived significantly shorter than patients with a low EGR3 fraction. ( F) The same trend was found in EGR3 mRNA data from TCGA, although results were non-significant . G) When sub-stratifying patients from the EGR1 high group by EGR3 expression, the group with high EGR3 expression had significantly shorter survival compared to patients with low EGR3 expression. H) Sub-stratification of patients from the EGR1 low group by EGR3 expression did not result in survival differences between the two groups. HR = Hazard ratio.

    Techniques Used: Expressing, Methylation

    Multivariate Cox-regression for glioblastoma patients.
    Figure Legend Snippet: Multivariate Cox-regression for glioblastoma patients.

    Techniques Used: Methylation, Mutagenesis

    MGMT-methylation distribution and multivariate cox-regression for EGR1/EGR3 combinations. (A) The distribution of MGMT-methylation status was highly skewed when looking at the two EGR1 groups: The majority of patients in the EGR1 high group had a methylated MGMT-promoter, while 100% of patients in the EGR1 low group had un-methylated MGMT-promoters. ( B) TCGA mRNA data showed a significant inverse correlation between EGR1 and MGMT mRNA levels. ( C) TCGA data showed that patients with recurrent GBM and high EGR3 mRNA levels had significantly shorter survival compared to patients with low EGR3 mRNA levels. ( D,E) Multivariate Cox-regression of the different EGR1 and EGR3 combinations showed that patients with EGR1 high/EGR3 high had a worse prognosis than patients with EGR1 high/EGR3 low. * = P < 0.05. ** = P < 0.01. *** = P < 0.001.
    Figure Legend Snippet: MGMT-methylation distribution and multivariate cox-regression for EGR1/EGR3 combinations. (A) The distribution of MGMT-methylation status was highly skewed when looking at the two EGR1 groups: The majority of patients in the EGR1 high group had a methylated MGMT-promoter, while 100% of patients in the EGR1 low group had un-methylated MGMT-promoters. ( B) TCGA mRNA data showed a significant inverse correlation between EGR1 and MGMT mRNA levels. ( C) TCGA data showed that patients with recurrent GBM and high EGR3 mRNA levels had significantly shorter survival compared to patients with low EGR3 mRNA levels. ( D,E) Multivariate Cox-regression of the different EGR1 and EGR3 combinations showed that patients with EGR1 high/EGR3 high had a worse prognosis than patients with EGR1 high/EGR3 low. * = P < 0.05. ** = P < 0.01. *** = P < 0.001.

    Techniques Used: Methylation

    egr3  (Cell Signaling Technology Inc)


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    Cell Signaling Technology Inc egr3
    Egr3, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    gfp  (Cell Signaling Technology Inc)


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    Cell Signaling Technology Inc gfp
    FBW7α targets SOX9 for ubiquitination and destruction after UV-induced DNA damage. ( A ) Screen of <t>F-box</t> <t>proteins.</t> H293T cells were co-transfected with FLAG-SOX9 and an empty vector or the GST-tagged F-box proteins FBXL3a, FBXL13, FBXL18, FBXO16, FBW4, FBW6 or FBW7, as well as green fluorescent protein <t>(GFP)</t> for a transfection control. Lysates were immunoblotted for FLAG and GST. α-Tubulin (α-TUB) was used as a loading control. ( B ) Western blot of FLAG-SOX9, HA-FBW7 and GFP of lysates from U2OS and H1299 cells that were co-transfected with FLAG-SOX9, GFP and increasing amounts of HA-FBW7α. α-tubulin was used as a loading control. ( C ) FBW7 mediates UV-induced SOX9 degradation. FBW7 +/+ or FBW 7 −/− HCT116 colon cancer cells UV irradiated (8 h, 80 J/m 2 ), or unirradiated, were immunoblotted for SOX9. GAPDH was used as a loading control. ( D ) FBW7 decreases endogenous SOX9. FBW7 −/− HCT116 were transfected with HA-FBW7. FBW7 +/+ and FBW7 −/− HCT116 cells were immunoblotted for SOX9 and HA-FBW7. GAPDH was used as a loading control. ( E ) RNA from FBW7 +/+ and FBW7 −/− HCT116 cells were examined for deletion of exon 5 in FBW7, utilizing primers that span exons 4 and 6 and that yield a 251 bp product. The absence of an RT-PCR product in lane 2 signifies deletion of exon 5 and de-stabilization of the mRNA. GAPDH was used as a control for mRNA quality.
    Gfp, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    1) Product Images from "SOX9 is targeted for proteasomal degradation by the E3 ligase FBW7 in response to DNA damage"

    Article Title: SOX9 is targeted for proteasomal degradation by the E3 ligase FBW7 in response to DNA damage

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkw748

    FBW7α targets SOX9 for ubiquitination and destruction after UV-induced DNA damage. ( A ) Screen of F-box proteins. H293T cells were co-transfected with FLAG-SOX9 and an empty vector or the GST-tagged F-box proteins FBXL3a, FBXL13, FBXL18, FBXO16, FBW4, FBW6 or FBW7, as well as green fluorescent protein (GFP) for a transfection control. Lysates were immunoblotted for FLAG and GST. α-Tubulin (α-TUB) was used as a loading control. ( B ) Western blot of FLAG-SOX9, HA-FBW7 and GFP of lysates from U2OS and H1299 cells that were co-transfected with FLAG-SOX9, GFP and increasing amounts of HA-FBW7α. α-tubulin was used as a loading control. ( C ) FBW7 mediates UV-induced SOX9 degradation. FBW7 +/+ or FBW 7 −/− HCT116 colon cancer cells UV irradiated (8 h, 80 J/m 2 ), or unirradiated, were immunoblotted for SOX9. GAPDH was used as a loading control. ( D ) FBW7 decreases endogenous SOX9. FBW7 −/− HCT116 were transfected with HA-FBW7. FBW7 +/+ and FBW7 −/− HCT116 cells were immunoblotted for SOX9 and HA-FBW7. GAPDH was used as a loading control. ( E ) RNA from FBW7 +/+ and FBW7 −/− HCT116 cells were examined for deletion of exon 5 in FBW7, utilizing primers that span exons 4 and 6 and that yield a 251 bp product. The absence of an RT-PCR product in lane 2 signifies deletion of exon 5 and de-stabilization of the mRNA. GAPDH was used as a control for mRNA quality.
    Figure Legend Snippet: FBW7α targets SOX9 for ubiquitination and destruction after UV-induced DNA damage. ( A ) Screen of F-box proteins. H293T cells were co-transfected with FLAG-SOX9 and an empty vector or the GST-tagged F-box proteins FBXL3a, FBXL13, FBXL18, FBXO16, FBW4, FBW6 or FBW7, as well as green fluorescent protein (GFP) for a transfection control. Lysates were immunoblotted for FLAG and GST. α-Tubulin (α-TUB) was used as a loading control. ( B ) Western blot of FLAG-SOX9, HA-FBW7 and GFP of lysates from U2OS and H1299 cells that were co-transfected with FLAG-SOX9, GFP and increasing amounts of HA-FBW7α. α-tubulin was used as a loading control. ( C ) FBW7 mediates UV-induced SOX9 degradation. FBW7 +/+ or FBW 7 −/− HCT116 colon cancer cells UV irradiated (8 h, 80 J/m 2 ), or unirradiated, were immunoblotted for SOX9. GAPDH was used as a loading control. ( D ) FBW7 decreases endogenous SOX9. FBW7 −/− HCT116 were transfected with HA-FBW7. FBW7 +/+ and FBW7 −/− HCT116 cells were immunoblotted for SOX9 and HA-FBW7. GAPDH was used as a loading control. ( E ) RNA from FBW7 +/+ and FBW7 −/− HCT116 cells were examined for deletion of exon 5 in FBW7, utilizing primers that span exons 4 and 6 and that yield a 251 bp product. The absence of an RT-PCR product in lane 2 signifies deletion of exon 5 and de-stabilization of the mRNA. GAPDH was used as a control for mRNA quality.

    Techniques Used: Transfection, Plasmid Preparation, Western Blot, Irradiation, Reverse Transcription Polymerase Chain Reaction

    UV-induced SOX9 degradation requires GSK3 activity. ( A ) GSK3 enhances FBW7α-mediated SOX9 degradation. H1299 cells were co-transfected with HA-FBW7α and FLAG-SOX9, and were either untreated or treated with CHIR-99021 in the presence of CHX. Lysates were collected at varying times and probed for FLAG-SOX9 and HA-FBW7. The graph on the right depicts the half-life of SOX9, determined by densitometric quantitation. GAPDH was used as a normalization control. SE, short film exposure; LE, long film exposure. ( B ) H1299 cells were transfected with FLAG-SOX9 with or without wild-type (WT) GSK3β or constitutively active S9A GSK3β and with or without HA-FBW7α, which was transfected at a 1:4 molar ratio to the other plasmids. GFP was transfected as a control. Lysates were immunoblotted for FLAG-SOX9, HA-FBW7, GFP and GSK3β. Levels of FLAG-SOX9 were quantified by densitometry, normalized to GAPDH. ( C ) FBW7α interacts with SOX9. H1299 and U2OS cells were transfected with HA-FBW7α along with FLAG-SOX9. Lysates were immunoprecipitated with anti-FLAG and immunoblotted for FLAG-SOX9 and HA-FBW7. A Western blot of the 5% input lysates is also shown ( D ) Endogenous FBW7 interacts with endogenous SOX9. SOX9 was immunoprecipitated (IP) from H1299 lysates, and proteins were immunoblotted for FBW7 and SOX9, as well as IgG. A Western blot of the 5% input lysates is also shown. ( E ) Interaction between FBW7α and SOX9 is dependent on GSK3. H1299 cells were transfected with HA-FBW7α along with FLAG-SOX9 and GFP as a transfection control, either untreated or treated with the GSK3 inhibitor, CHIR99021. Lysates were immunoprecipitated with anti-FLAG and immunoblotted for FLAG-SOX9 and HA-FBW7. A Western blot of the 5% input lysates is also shown. ( F ) FBW7 targets SOX9 for ubiquitination. H1299 cells were co-transfected with either FLAG-SOX9 or FLAG-MUT-SOX9 (CPD-mutant), and HA-Ub with or without HA-FBW7α. Cells were treated with MG132 with or without CHIR99021 for 6 h before collection of lysates. GFP was transfected as a control. Denatured lysates were immunoprecipitated with anti-FLAG and immunoblotted for Ubiquitin. The 5% input lysates were also immunoblotted for HA, FLAG and GFP.
    Figure Legend Snippet: UV-induced SOX9 degradation requires GSK3 activity. ( A ) GSK3 enhances FBW7α-mediated SOX9 degradation. H1299 cells were co-transfected with HA-FBW7α and FLAG-SOX9, and were either untreated or treated with CHIR-99021 in the presence of CHX. Lysates were collected at varying times and probed for FLAG-SOX9 and HA-FBW7. The graph on the right depicts the half-life of SOX9, determined by densitometric quantitation. GAPDH was used as a normalization control. SE, short film exposure; LE, long film exposure. ( B ) H1299 cells were transfected with FLAG-SOX9 with or without wild-type (WT) GSK3β or constitutively active S9A GSK3β and with or without HA-FBW7α, which was transfected at a 1:4 molar ratio to the other plasmids. GFP was transfected as a control. Lysates were immunoblotted for FLAG-SOX9, HA-FBW7, GFP and GSK3β. Levels of FLAG-SOX9 were quantified by densitometry, normalized to GAPDH. ( C ) FBW7α interacts with SOX9. H1299 and U2OS cells were transfected with HA-FBW7α along with FLAG-SOX9. Lysates were immunoprecipitated with anti-FLAG and immunoblotted for FLAG-SOX9 and HA-FBW7. A Western blot of the 5% input lysates is also shown ( D ) Endogenous FBW7 interacts with endogenous SOX9. SOX9 was immunoprecipitated (IP) from H1299 lysates, and proteins were immunoblotted for FBW7 and SOX9, as well as IgG. A Western blot of the 5% input lysates is also shown. ( E ) Interaction between FBW7α and SOX9 is dependent on GSK3. H1299 cells were transfected with HA-FBW7α along with FLAG-SOX9 and GFP as a transfection control, either untreated or treated with the GSK3 inhibitor, CHIR99021. Lysates were immunoprecipitated with anti-FLAG and immunoblotted for FLAG-SOX9 and HA-FBW7. A Western blot of the 5% input lysates is also shown. ( F ) FBW7 targets SOX9 for ubiquitination. H1299 cells were co-transfected with either FLAG-SOX9 or FLAG-MUT-SOX9 (CPD-mutant), and HA-Ub with or without HA-FBW7α. Cells were treated with MG132 with or without CHIR99021 for 6 h before collection of lysates. GFP was transfected as a control. Denatured lysates were immunoprecipitated with anti-FLAG and immunoblotted for Ubiquitin. The 5% input lysates were also immunoblotted for HA, FLAG and GFP.

    Techniques Used: Activity Assay, Transfection, Quantitation Assay, Immunoprecipitation, Western Blot, Mutagenesis

    Phosphorylation of SOX9 in the K2 domain by GSK3β triggers SOX9 destruction by FBW7α. ( A ) Alignment of the putative SOX9 consensus phosphodegron (CPD) sites (Motifs 1–4) with the FBW7 CPD sequence known for other FBW7 target proteins. The SOX9 serine and threonine residues that were mutated to alanine are underlined. ( B ) The FBW7α CPD binding site on SOX9 comprises amino acids 236–240. The top schematic depicts the locations of the DIM, HMG, K2, PQA and TA domains of SOX9, as well as the locations of the putative CPD motifs. Below, H1299 cells were transfected with WT FLAG-SOX9 or the indicated FLAG-SOX9 mutants (M1–M4) with GFP and with or without HA-FBW7α. Lysates were immunoblotted for GFP, FLAG and HA. GAPDH was used as a normalization control. SE, short film exposure; LE, long film exposure. ( C ) The SOX9 CPD sequence targeted by FBW7α is conserved across different species. The sequence shown for zebrafish is the SOX9a ortholog. ( D ) SOX9 interacts with FBW7α at the K2 domain CPD motif. H1299 cells were co-transfected with HA-FBW7α and either wild-type FLAG-SOX9 or CPD-mutant FLAG-MUT-SOX9. Lysates were immunoprecipitated with anti-FLAG and immunoblotted for HA and FLAG. A Western blot of lysates comprising 5% of input is also shown for HA and FLAG. ( E ) The SOX9 K2 domain is phosphorylated by GSK3β at T236. Beas2b cells were transfected with the FLAG-control, wild-type FLAG-SOX9 or CPD-mutant FLAG-MUT-SOX9. Cells were treated with 40 μM CHIR-99021 for 8 h, 48 h after transfection. A Western blot of lysates is shown for SOX9, P-T236-SOX9 and GAPDH. SE, short film exposure; LE, long film exposure. ( F ) FBW7α directly interacts with SOX9 in vitro . A binding assay was performed on HA-FBW7α protein and a GST-tagged WT SOX9 fragment comprising amino acids 186–318, or a GST-tagged SOX9 mutated at the CPD motif, with or without human recombinant GSK3β (rhGSK3β). The input and GST-immunoprecipitated proteins were immunoblotted for HA-FBW7. Total GST and GST-SOX9 were visualized by a coomassie blue stain. M, protein marker. ( G ) GSK3β directly phosphorylates SOX9. An in vitro kinase assay was performed using human recombinant GSK3β and purified GST, wild-type GST-SOX9 or a CPD-mutant GST-SOX9. 32 P GST-SOX9 is shown. Total GST and GST-SOX9 were visualized by a coomassie blue stain. Presence of GSK3β was confirmed by immunoblotting. Graph represents the average phosphorylation of CPD-mutant SOX relative to wild-type SOX9 in three independent experiments. Error bars represent the standard deviation. A two-tailed Student's t -test was used to compare groups. **, P < 0.01.
    Figure Legend Snippet: Phosphorylation of SOX9 in the K2 domain by GSK3β triggers SOX9 destruction by FBW7α. ( A ) Alignment of the putative SOX9 consensus phosphodegron (CPD) sites (Motifs 1–4) with the FBW7 CPD sequence known for other FBW7 target proteins. The SOX9 serine and threonine residues that were mutated to alanine are underlined. ( B ) The FBW7α CPD binding site on SOX9 comprises amino acids 236–240. The top schematic depicts the locations of the DIM, HMG, K2, PQA and TA domains of SOX9, as well as the locations of the putative CPD motifs. Below, H1299 cells were transfected with WT FLAG-SOX9 or the indicated FLAG-SOX9 mutants (M1–M4) with GFP and with or without HA-FBW7α. Lysates were immunoblotted for GFP, FLAG and HA. GAPDH was used as a normalization control. SE, short film exposure; LE, long film exposure. ( C ) The SOX9 CPD sequence targeted by FBW7α is conserved across different species. The sequence shown for zebrafish is the SOX9a ortholog. ( D ) SOX9 interacts with FBW7α at the K2 domain CPD motif. H1299 cells were co-transfected with HA-FBW7α and either wild-type FLAG-SOX9 or CPD-mutant FLAG-MUT-SOX9. Lysates were immunoprecipitated with anti-FLAG and immunoblotted for HA and FLAG. A Western blot of lysates comprising 5% of input is also shown for HA and FLAG. ( E ) The SOX9 K2 domain is phosphorylated by GSK3β at T236. Beas2b cells were transfected with the FLAG-control, wild-type FLAG-SOX9 or CPD-mutant FLAG-MUT-SOX9. Cells were treated with 40 μM CHIR-99021 for 8 h, 48 h after transfection. A Western blot of lysates is shown for SOX9, P-T236-SOX9 and GAPDH. SE, short film exposure; LE, long film exposure. ( F ) FBW7α directly interacts with SOX9 in vitro . A binding assay was performed on HA-FBW7α protein and a GST-tagged WT SOX9 fragment comprising amino acids 186–318, or a GST-tagged SOX9 mutated at the CPD motif, with or without human recombinant GSK3β (rhGSK3β). The input and GST-immunoprecipitated proteins were immunoblotted for HA-FBW7. Total GST and GST-SOX9 were visualized by a coomassie blue stain. M, protein marker. ( G ) GSK3β directly phosphorylates SOX9. An in vitro kinase assay was performed using human recombinant GSK3β and purified GST, wild-type GST-SOX9 or a CPD-mutant GST-SOX9. 32 P GST-SOX9 is shown. Total GST and GST-SOX9 were visualized by a coomassie blue stain. Presence of GSK3β was confirmed by immunoblotting. Graph represents the average phosphorylation of CPD-mutant SOX relative to wild-type SOX9 in three independent experiments. Error bars represent the standard deviation. A two-tailed Student's t -test was used to compare groups. **, P < 0.01.

    Techniques Used: Sequencing, Binding Assay, Transfection, Mutagenesis, Immunoprecipitation, Western Blot, In Vitro, Recombinant, Staining, Marker, Kinase Assay, Purification, Standard Deviation, Two Tailed Test

    egr 3 antibodies  (Cell Signaling Technology Inc)


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    Cell Signaling Technology Inc egr 3 antibodies
    Egr 3 Antibodies, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Egr 3, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Cell Signaling Technology Inc egr3 antibody
    <t>EGR3</t> expression in human prostate cell lines. ( A ) The EGR3 gene expression values were retrieved from GEO data set GDS3155 that contains expression data for 15 human prostate epithelial cell lines (note that the stroma cell line WPMY present in the data set was excluded from this figure). ( B ) Box plot of the mRNA expression of EGR family members in this data set, reflecting high variability (EGR1) and low variability of expression (EGR2–4). ( C ) EGR3 protein expression in a few prostate cell lines. Untreated cells were lysed and protein expression was analysed by western blot using anti-EGR3 antibodies. The EGR3 protein migrates at ∼50 kDa. Membranes were reprobed with anti-actin antibodies as loading control. ( D ) Comparison of EGR3 mRNA expression in M12 and P69 cells. Total RNA was extracted from untreated cells and EGR3 expression was measured by quantitative RT–PCR. Results are expressed relative to expression in P69 cells. ( E ) Subcellular localisation of EGR3 in M12 prostate cancer cells. Untreated prostate cancer cells M12 were lysed and fractionated using a commercial kit, followed by western blot analysis of protein levels using anti-EGR3 antibodies.
    Egr3 Antibody, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    1) Product Images from "Early Growth Response 3 regulates genes of inflammation and directly activates IL6 and IL8 expression in prostate cancer"

    Article Title: Early Growth Response 3 regulates genes of inflammation and directly activates IL6 and IL8 expression in prostate cancer

    Journal: British Journal of Cancer

    doi: 10.1038/bjc.2014.622

    EGR3 expression in human prostate cell lines. ( A ) The EGR3 gene expression values were retrieved from GEO data set GDS3155 that contains expression data for 15 human prostate epithelial cell lines (note that the stroma cell line WPMY present in the data set was excluded from this figure). ( B ) Box plot of the mRNA expression of EGR family members in this data set, reflecting high variability (EGR1) and low variability of expression (EGR2–4). ( C ) EGR3 protein expression in a few prostate cell lines. Untreated cells were lysed and protein expression was analysed by western blot using anti-EGR3 antibodies. The EGR3 protein migrates at ∼50 kDa. Membranes were reprobed with anti-actin antibodies as loading control. ( D ) Comparison of EGR3 mRNA expression in M12 and P69 cells. Total RNA was extracted from untreated cells and EGR3 expression was measured by quantitative RT–PCR. Results are expressed relative to expression in P69 cells. ( E ) Subcellular localisation of EGR3 in M12 prostate cancer cells. Untreated prostate cancer cells M12 were lysed and fractionated using a commercial kit, followed by western blot analysis of protein levels using anti-EGR3 antibodies.
    Figure Legend Snippet: EGR3 expression in human prostate cell lines. ( A ) The EGR3 gene expression values were retrieved from GEO data set GDS3155 that contains expression data for 15 human prostate epithelial cell lines (note that the stroma cell line WPMY present in the data set was excluded from this figure). ( B ) Box plot of the mRNA expression of EGR family members in this data set, reflecting high variability (EGR1) and low variability of expression (EGR2–4). ( C ) EGR3 protein expression in a few prostate cell lines. Untreated cells were lysed and protein expression was analysed by western blot using anti-EGR3 antibodies. The EGR3 protein migrates at ∼50 kDa. Membranes were reprobed with anti-actin antibodies as loading control. ( D ) Comparison of EGR3 mRNA expression in M12 and P69 cells. Total RNA was extracted from untreated cells and EGR3 expression was measured by quantitative RT–PCR. Results are expressed relative to expression in P69 cells. ( E ) Subcellular localisation of EGR3 in M12 prostate cancer cells. Untreated prostate cancer cells M12 were lysed and fractionated using a commercial kit, followed by western blot analysis of protein levels using anti-EGR3 antibodies.

    Techniques Used: Expressing, Western Blot, Quantitative RT-PCR

    Inducibility of EGR3 in M12 and P69 prostate epithelial cells. Cells were treated with TPA (10 ng ml −1 ) or FBS (10%) for the indicated times. The protein expression levels of EGR3 and EGR1 were measured by western blot using successively EGR3 and EGR1 antibodies (without stripping). The EGR1 was used as a positive control for the effect of TPA and FBS on the expression of early response genes. Membranes were then stripped and reprobed with anti-actin antibodies as loading control. ( A ) P69 cells. The symbol (*) denotes the position of EGR3 if it was present. ( B ) M12 cells. ( C ) Conditioned medium (CM) from M12 cells was applied to P69 cells for the indicated times. The P69 cells were lysed and EGR3 protein levels were measured by western blot. Cell lysates from untreated M12 cells were used as positive control (far right lane). Note that the signal intensity for EGR3 is different than in other western blots because a different commercial antibody was used.
    Figure Legend Snippet: Inducibility of EGR3 in M12 and P69 prostate epithelial cells. Cells were treated with TPA (10 ng ml −1 ) or FBS (10%) for the indicated times. The protein expression levels of EGR3 and EGR1 were measured by western blot using successively EGR3 and EGR1 antibodies (without stripping). The EGR1 was used as a positive control for the effect of TPA and FBS on the expression of early response genes. Membranes were then stripped and reprobed with anti-actin antibodies as loading control. ( A ) P69 cells. The symbol (*) denotes the position of EGR3 if it was present. ( B ) M12 cells. ( C ) Conditioned medium (CM) from M12 cells was applied to P69 cells for the indicated times. The P69 cells were lysed and EGR3 protein levels were measured by western blot. Cell lysates from untreated M12 cells were used as positive control (far right lane). Note that the signal intensity for EGR3 is different than in other western blots because a different commercial antibody was used.

    Techniques Used: Expressing, Western Blot, Stripping Membranes, Positive Control

    The shRNA-mediated EGR3 silencing in M12 cells. ( A ) The M12 cells were stably transfected with the empty vector, a scramble control shRNA (shSCR-M12), or EGR3-shRNA (shEGR3-clone 2 and shEGR3-clone 3). Protein expression of EGR3 was analysed by western blotting at various cell passages. A representative western blot is shown. ( B ) Western blots from three separate experiments (at three different cell culture passages) were quantified using ImageJ processing software. The graph represents the average of EGR3 expression compared with shSCR±S.E.
    Figure Legend Snippet: The shRNA-mediated EGR3 silencing in M12 cells. ( A ) The M12 cells were stably transfected with the empty vector, a scramble control shRNA (shSCR-M12), or EGR3-shRNA (shEGR3-clone 2 and shEGR3-clone 3). Protein expression of EGR3 was analysed by western blotting at various cell passages. A representative western blot is shown. ( B ) Western blots from three separate experiments (at three different cell culture passages) were quantified using ImageJ processing software. The graph represents the average of EGR3 expression compared with shSCR±S.E.

    Techniques Used: shRNA, Stable Transfection, Transfection, Plasmid Preparation, Expressing, Western Blot, Cell Culture, Software

    Top 20 genes that are induced by  EGR3  in M12 cells (i.e., genes for which expression was lower in shEGR3-M12-silenced cells compared with shSCR-M12 control cells)
    Figure Legend Snippet: Top 20 genes that are induced by EGR3 in M12 cells (i.e., genes for which expression was lower in shEGR3-M12-silenced cells compared with shSCR-M12 control cells)

    Techniques Used: Expressing, Significance Assay, Activity Assay, Binding Assay

    Top 20 genes that are repressed by  EGR3  in M12 cells (i.e., genes for which expression was higher in shEGR3-M12-silenced cells compared with shSCR-M12 control cells)
    Figure Legend Snippet: Top 20 genes that are repressed by EGR3 in M12 cells (i.e., genes for which expression was higher in shEGR3-M12-silenced cells compared with shSCR-M12 control cells)

    Techniques Used: Expressing, Significance Assay, Binding Assay

    GeneGo enrichment of  EGR3-regulated  genes
    Figure Legend Snippet: GeneGo enrichment of EGR3-regulated genes

    Techniques Used: Significance Assay, Infection, Coagulation

    Early Growth Response 3 regulates Il6 and IL8 in M12 prostate cancer cells. ( A ) A Venn diagram compares the list of genes that were induced by EGR3 in M12 cells with the list of genes that were highly correlated with EGR3 in clinical human prostate cancer samples. The list of nine genes found at the intersection is shown below the diagram. ( B ) Confirmation of EGR3-mediated regulation of gene expression. Total RNA was extracted from stable cell lines control (shSCR-M12) and shEGR3 (shEGR3-cl2 and shEGR3-cl3). The mRNA expression was analysed by quantitative RT–PCR using specific primers. The housekeeping gene GAPDH was used as reference. Expression values obtained for each shEGR3 clone were compared with shSCR control using the 2 −ΔΔ CT method. ( C ) Chromatin immunoprecipitation of EGR3 target genes IL6 and IL8 . Cells fixed with paraformaldehyde and a specific EGR3 antibody was used to capture protein–DNA complexes. Nonimmune IgG was used as negative control. Promoter regions were amplified by real-time qPCR and analysed by electrophoresis on agarose gels.
    Figure Legend Snippet: Early Growth Response 3 regulates Il6 and IL8 in M12 prostate cancer cells. ( A ) A Venn diagram compares the list of genes that were induced by EGR3 in M12 cells with the list of genes that were highly correlated with EGR3 in clinical human prostate cancer samples. The list of nine genes found at the intersection is shown below the diagram. ( B ) Confirmation of EGR3-mediated regulation of gene expression. Total RNA was extracted from stable cell lines control (shSCR-M12) and shEGR3 (shEGR3-cl2 and shEGR3-cl3). The mRNA expression was analysed by quantitative RT–PCR using specific primers. The housekeeping gene GAPDH was used as reference. Expression values obtained for each shEGR3 clone were compared with shSCR control using the 2 −ΔΔ CT method. ( C ) Chromatin immunoprecipitation of EGR3 target genes IL6 and IL8 . Cells fixed with paraformaldehyde and a specific EGR3 antibody was used to capture protein–DNA complexes. Nonimmune IgG was used as negative control. Promoter regions were amplified by real-time qPCR and analysed by electrophoresis on agarose gels.

    Techniques Used: Expressing, Stable Transfection, Quantitative RT-PCR, Chromatin Immunoprecipitation, Negative Control, Amplification, Electrophoresis

    Forced expression of EGR3 induces IL6 and IL8 in P69 cells. ( A ) Early Growth Response 3 transfection in P69 cells. Cells were transfected with a tag-EGR3 expression plasmid or an empty plasmid as described in the Materials and Methods. Transfected cells were lysed and EGR3 protein expression was analysed by western blotting using antibodies against the V5 tag. ( B ) Subcellular localisation of EGR3 in transfected P69 cells. Untreated cells were lysed and fractionated using a commercial kit, followed by western blotting. ( C ) Total RNA was extracted from control or EGR3-expressing P69 cells and the mRNA expression of various EGR3-correlated genes was analysed by real-time qPCR. The housekeeping gene GAPDH was used as reference. Expression values were compared using the 2 −ΔΔ CT method.
    Figure Legend Snippet: Forced expression of EGR3 induces IL6 and IL8 in P69 cells. ( A ) Early Growth Response 3 transfection in P69 cells. Cells were transfected with a tag-EGR3 expression plasmid or an empty plasmid as described in the Materials and Methods. Transfected cells were lysed and EGR3 protein expression was analysed by western blotting using antibodies against the V5 tag. ( B ) Subcellular localisation of EGR3 in transfected P69 cells. Untreated cells were lysed and fractionated using a commercial kit, followed by western blotting. ( C ) Total RNA was extracted from control or EGR3-expressing P69 cells and the mRNA expression of various EGR3-correlated genes was analysed by real-time qPCR. The housekeeping gene GAPDH was used as reference. Expression values were compared using the 2 −ΔΔ CT method.

    Techniques Used: Expressing, Transfection, Plasmid Preparation, Western Blot

    egr3  (Cell Signaling Technology Inc)


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    Cell Signaling Technology Inc egr3
    In A , shown is a Western blot analysis of primary esophageal epithelial cells pre-treated with IL-13 for 24 hr and then treated with recombinant human NGF for 0, 5, or 15 min. pNTRK1 indicates phosphorylated protein (arrow). In B , shown is Western blot analysis of TE-7 cells pre-treated with IL-13 for 24 hr and then treated with NGF for 5 min. pNTRK1 and pERK1/2 indicate phosphorylated proteins, arrow points at pNTRK1. In C , the kinetics of NTRK1 phosphorylation were assessed by Western blot. Cells were pre-treated with IL-13 for 24 hr and then treated with NGF for 5, 15 or 30 min. For A–C , phosphorylation was assessed at tyrosine residues Tyr674/675 in the catalytic domain of NTRK1. In D , kinetic analysis of EGR1 and <t>EGR3</t> mRNA in TE-7 cells pre-treated with IL-13 for 24 hr followed by treatment with NGF for 1, 2, or 6 hrs was performed by RT-PCR. Fold change indicates increase over untreated (no IL-13) cells stimulated with NGF (+NGF). NGF was used at the concentration of 100 ng/ml. Data for 3 independent experiments are presented as mean value with standard error measurements; ****p < 0.0001, *p < 0.05. In E , EGR1 and EGR3 protein levels in TE-7 cells pre-treated with IL-13 for 24 hr followed by treatment with NGF were analyzed by Western blot; p38 serves as a loading control.
    Egr3, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    1) Product Images from "Neurotrophic tyrosine kinase receptor 1 is a direct transcriptional and epigenetic target of IL-13 involved in allergic inflammation"

    Article Title: Neurotrophic tyrosine kinase receptor 1 is a direct transcriptional and epigenetic target of IL-13 involved in allergic inflammation

    Journal: Mucosal immunology

    doi: 10.1038/mi.2014.109

    In A , shown is a Western blot analysis of primary esophageal epithelial cells pre-treated with IL-13 for 24 hr and then treated with recombinant human NGF for 0, 5, or 15 min. pNTRK1 indicates phosphorylated protein (arrow). In B , shown is Western blot analysis of TE-7 cells pre-treated with IL-13 for 24 hr and then treated with NGF for 5 min. pNTRK1 and pERK1/2 indicate phosphorylated proteins, arrow points at pNTRK1. In C , the kinetics of NTRK1 phosphorylation were assessed by Western blot. Cells were pre-treated with IL-13 for 24 hr and then treated with NGF for 5, 15 or 30 min. For A–C , phosphorylation was assessed at tyrosine residues Tyr674/675 in the catalytic domain of NTRK1. In D , kinetic analysis of EGR1 and EGR3 mRNA in TE-7 cells pre-treated with IL-13 for 24 hr followed by treatment with NGF for 1, 2, or 6 hrs was performed by RT-PCR. Fold change indicates increase over untreated (no IL-13) cells stimulated with NGF (+NGF). NGF was used at the concentration of 100 ng/ml. Data for 3 independent experiments are presented as mean value with standard error measurements; ****p < 0.0001, *p < 0.05. In E , EGR1 and EGR3 protein levels in TE-7 cells pre-treated with IL-13 for 24 hr followed by treatment with NGF were analyzed by Western blot; p38 serves as a loading control.
    Figure Legend Snippet: In A , shown is a Western blot analysis of primary esophageal epithelial cells pre-treated with IL-13 for 24 hr and then treated with recombinant human NGF for 0, 5, or 15 min. pNTRK1 indicates phosphorylated protein (arrow). In B , shown is Western blot analysis of TE-7 cells pre-treated with IL-13 for 24 hr and then treated with NGF for 5 min. pNTRK1 and pERK1/2 indicate phosphorylated proteins, arrow points at pNTRK1. In C , the kinetics of NTRK1 phosphorylation were assessed by Western blot. Cells were pre-treated with IL-13 for 24 hr and then treated with NGF for 5, 15 or 30 min. For A–C , phosphorylation was assessed at tyrosine residues Tyr674/675 in the catalytic domain of NTRK1. In D , kinetic analysis of EGR1 and EGR3 mRNA in TE-7 cells pre-treated with IL-13 for 24 hr followed by treatment with NGF for 1, 2, or 6 hrs was performed by RT-PCR. Fold change indicates increase over untreated (no IL-13) cells stimulated with NGF (+NGF). NGF was used at the concentration of 100 ng/ml. Data for 3 independent experiments are presented as mean value with standard error measurements; ****p < 0.0001, *p < 0.05. In E , EGR1 and EGR3 protein levels in TE-7 cells pre-treated with IL-13 for 24 hr followed by treatment with NGF were analyzed by Western blot; p38 serves as a loading control.

    Techniques Used: Western Blot, Recombinant, Reverse Transcription Polymerase Chain Reaction, Concentration Assay

    In A , a representative immunofluorescent image of TE-7 cells infected with either empty virus (control pool) or virus expressing NTRK1 (NTRK1 pool) is shown. In B , shown is Western blot analysis of two independent pools of TE-7 cells stably expressing NTRK1 (NTRK1 (1) and NTRK1 (2)). Cells were treated with NGF (100 ng/ml) for the indicated times. pNTRK1 and pERK1/2 indicate phosphorylated proteins. In C , the effect of NGF and IL-13 stimulation on EGR1 , EGR3 and CCL26 expression was assessed by RT-PCR in control (pLVX Ctrl) and NTRK1 stably expressing cells (pLVX NTRK1). In D , effect of NTRK1 down regulation in TE-7 cells stably expressing NTRK1 on EGR1 and EGR3 induction by NGF was assessed by RT-PCR. Cells were treated with siRNA for 36 hr followed by stimulated with NGF at 10 ng/ml for indicated periods of time. Expression level of EGRs relative to siCtrl-treated cells is shown. Data for 3 independent experiments are presented; error bars represent standard error measurements In E , the effect of decreased expression of NTRK1 in IL-13—pretreated TE-7 control pool on EGR1 and EGR3 induction by NGF was assessed by RT-PCR. Cells were treated with siRNA for 18 hr, induced with IL-13 at 10 ng/ml for 24 hr followed by stimulation with NGF at 10 ng/ml for 1 hr. siCtrl – control siRNA, siNTRK1 – siRNA against NTRK1. Data for 3 independent experiments are presented as box and whiskers plot. In F , the effect of pre-treatment of TE-7 cells stably expressing NTRK1 with tyrosine kinase inhibitors on EGR1 and EGR3 induction by NGF was assessed by RT-PCR. Cells were pre-treated with the inhibitors at 0.1 μM for 15 min following stimulation with NGF at 10 ng/ml for 2 hr. Data for 2 independent experiments are shown. ****p < 0.0001, **p < 0.01, *p < 0.05, ns – not significant.
    Figure Legend Snippet: In A , a representative immunofluorescent image of TE-7 cells infected with either empty virus (control pool) or virus expressing NTRK1 (NTRK1 pool) is shown. In B , shown is Western blot analysis of two independent pools of TE-7 cells stably expressing NTRK1 (NTRK1 (1) and NTRK1 (2)). Cells were treated with NGF (100 ng/ml) for the indicated times. pNTRK1 and pERK1/2 indicate phosphorylated proteins. In C , the effect of NGF and IL-13 stimulation on EGR1 , EGR3 and CCL26 expression was assessed by RT-PCR in control (pLVX Ctrl) and NTRK1 stably expressing cells (pLVX NTRK1). In D , effect of NTRK1 down regulation in TE-7 cells stably expressing NTRK1 on EGR1 and EGR3 induction by NGF was assessed by RT-PCR. Cells were treated with siRNA for 36 hr followed by stimulated with NGF at 10 ng/ml for indicated periods of time. Expression level of EGRs relative to siCtrl-treated cells is shown. Data for 3 independent experiments are presented; error bars represent standard error measurements In E , the effect of decreased expression of NTRK1 in IL-13—pretreated TE-7 control pool on EGR1 and EGR3 induction by NGF was assessed by RT-PCR. Cells were treated with siRNA for 18 hr, induced with IL-13 at 10 ng/ml for 24 hr followed by stimulation with NGF at 10 ng/ml for 1 hr. siCtrl – control siRNA, siNTRK1 – siRNA against NTRK1. Data for 3 independent experiments are presented as box and whiskers plot. In F , the effect of pre-treatment of TE-7 cells stably expressing NTRK1 with tyrosine kinase inhibitors on EGR1 and EGR3 induction by NGF was assessed by RT-PCR. Cells were pre-treated with the inhibitors at 0.1 μM for 15 min following stimulation with NGF at 10 ng/ml for 2 hr. Data for 2 independent experiments are shown. ****p < 0.0001, **p < 0.01, *p < 0.05, ns – not significant.

    Techniques Used: Infection, Expressing, Western Blot, Stable Transfection, Reverse Transcription Polymerase Chain Reaction

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    Cell Signaling Technology Inc egr3
    (A)Immunoblots for Egr1, Egr2 and <t>Egr3</t> before or at the indicated time points after the stimulation with plate-bound anti-CD3ε and anti-CD28 antibodies in DP cells from the indicated genotypes. Actin was used as a loading control. Data are representative of three independent experiments. (B) Immunoblots for Egr1, Egr2 and Egr3 in DP cells from the indicated genotypes before or at the indicated time points after the stimulation with plate-bound anti-CD3ε and anti-CD28 antibodies under the condition with U0126 in DMSO or with DMSO alone. Actin was used as a loading control. Data are representative of three independent experiments. (C, D) Quantitative RT-PCR analysis of Egr1 , Egr2 and Egr3 expression before (C) or at the indicated time points after the stimulation with anti-CD3ε and anti-CD28 antibodies (D) in DP cells from Zfat f/f and Zfat f/f - LckCre mice. The relative expression for each gene was normalized by the expression of Actb . The results are presented as the value relative to the unstimulated DP cells from Zfat f/f mice. The data are the mean ± s.d.; n.s., not significant.
    Egr3, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Cell Signaling Technology Inc egr3 antibody
    Patient characteristics.
    Egr3 Antibody, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Cell Signaling Technology Inc gfp
    FBW7α targets SOX9 for ubiquitination and destruction after UV-induced DNA damage. ( A ) Screen of <t>F-box</t> <t>proteins.</t> H293T cells were co-transfected with FLAG-SOX9 and an empty vector or the GST-tagged F-box proteins FBXL3a, FBXL13, FBXL18, FBXO16, FBW4, FBW6 or FBW7, as well as green fluorescent protein <t>(GFP)</t> for a transfection control. Lysates were immunoblotted for FLAG and GST. α-Tubulin (α-TUB) was used as a loading control. ( B ) Western blot of FLAG-SOX9, HA-FBW7 and GFP of lysates from U2OS and H1299 cells that were co-transfected with FLAG-SOX9, GFP and increasing amounts of HA-FBW7α. α-tubulin was used as a loading control. ( C ) FBW7 mediates UV-induced SOX9 degradation. FBW7 +/+ or FBW 7 −/− HCT116 colon cancer cells UV irradiated (8 h, 80 J/m 2 ), or unirradiated, were immunoblotted for SOX9. GAPDH was used as a loading control. ( D ) FBW7 decreases endogenous SOX9. FBW7 −/− HCT116 were transfected with HA-FBW7. FBW7 +/+ and FBW7 −/− HCT116 cells were immunoblotted for SOX9 and HA-FBW7. GAPDH was used as a loading control. ( E ) RNA from FBW7 +/+ and FBW7 −/− HCT116 cells were examined for deletion of exon 5 in FBW7, utilizing primers that span exons 4 and 6 and that yield a 251 bp product. The absence of an RT-PCR product in lane 2 signifies deletion of exon 5 and de-stabilization of the mRNA. GAPDH was used as a control for mRNA quality.
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    Cell Signaling Technology Inc egr 3 antibodies
    FBW7α targets SOX9 for ubiquitination and destruction after UV-induced DNA damage. ( A ) Screen of <t>F-box</t> <t>proteins.</t> H293T cells were co-transfected with FLAG-SOX9 and an empty vector or the GST-tagged F-box proteins FBXL3a, FBXL13, FBXL18, FBXO16, FBW4, FBW6 or FBW7, as well as green fluorescent protein <t>(GFP)</t> for a transfection control. Lysates were immunoblotted for FLAG and GST. α-Tubulin (α-TUB) was used as a loading control. ( B ) Western blot of FLAG-SOX9, HA-FBW7 and GFP of lysates from U2OS and H1299 cells that were co-transfected with FLAG-SOX9, GFP and increasing amounts of HA-FBW7α. α-tubulin was used as a loading control. ( C ) FBW7 mediates UV-induced SOX9 degradation. FBW7 +/+ or FBW 7 −/− HCT116 colon cancer cells UV irradiated (8 h, 80 J/m 2 ), or unirradiated, were immunoblotted for SOX9. GAPDH was used as a loading control. ( D ) FBW7 decreases endogenous SOX9. FBW7 −/− HCT116 were transfected with HA-FBW7. FBW7 +/+ and FBW7 −/− HCT116 cells were immunoblotted for SOX9 and HA-FBW7. GAPDH was used as a loading control. ( E ) RNA from FBW7 +/+ and FBW7 −/− HCT116 cells were examined for deletion of exon 5 in FBW7, utilizing primers that span exons 4 and 6 and that yield a 251 bp product. The absence of an RT-PCR product in lane 2 signifies deletion of exon 5 and de-stabilization of the mRNA. GAPDH was used as a control for mRNA quality.
    Egr 3 Antibodies, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/egr 3 antibodies/product/Cell Signaling Technology Inc
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    Cell Signaling Technology Inc egr 3
    FBW7α targets SOX9 for ubiquitination and destruction after UV-induced DNA damage. ( A ) Screen of <t>F-box</t> <t>proteins.</t> H293T cells were co-transfected with FLAG-SOX9 and an empty vector or the GST-tagged F-box proteins FBXL3a, FBXL13, FBXL18, FBXO16, FBW4, FBW6 or FBW7, as well as green fluorescent protein <t>(GFP)</t> for a transfection control. Lysates were immunoblotted for FLAG and GST. α-Tubulin (α-TUB) was used as a loading control. ( B ) Western blot of FLAG-SOX9, HA-FBW7 and GFP of lysates from U2OS and H1299 cells that were co-transfected with FLAG-SOX9, GFP and increasing amounts of HA-FBW7α. α-tubulin was used as a loading control. ( C ) FBW7 mediates UV-induced SOX9 degradation. FBW7 +/+ or FBW 7 −/− HCT116 colon cancer cells UV irradiated (8 h, 80 J/m 2 ), or unirradiated, were immunoblotted for SOX9. GAPDH was used as a loading control. ( D ) FBW7 decreases endogenous SOX9. FBW7 −/− HCT116 were transfected with HA-FBW7. FBW7 +/+ and FBW7 −/− HCT116 cells were immunoblotted for SOX9 and HA-FBW7. GAPDH was used as a loading control. ( E ) RNA from FBW7 +/+ and FBW7 −/− HCT116 cells were examined for deletion of exon 5 in FBW7, utilizing primers that span exons 4 and 6 and that yield a 251 bp product. The absence of an RT-PCR product in lane 2 signifies deletion of exon 5 and de-stabilization of the mRNA. GAPDH was used as a control for mRNA quality.
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    (A)Immunoblots for Egr1, Egr2 and Egr3 before or at the indicated time points after the stimulation with plate-bound anti-CD3ε and anti-CD28 antibodies in DP cells from the indicated genotypes. Actin was used as a loading control. Data are representative of three independent experiments. (B) Immunoblots for Egr1, Egr2 and Egr3 in DP cells from the indicated genotypes before or at the indicated time points after the stimulation with plate-bound anti-CD3ε and anti-CD28 antibodies under the condition with U0126 in DMSO or with DMSO alone. Actin was used as a loading control. Data are representative of three independent experiments. (C, D) Quantitative RT-PCR analysis of Egr1 , Egr2 and Egr3 expression before (C) or at the indicated time points after the stimulation with anti-CD3ε and anti-CD28 antibodies (D) in DP cells from Zfat f/f and Zfat f/f - LckCre mice. The relative expression for each gene was normalized by the expression of Actb . The results are presented as the value relative to the unstimulated DP cells from Zfat f/f mice. The data are the mean ± s.d.; n.s., not significant.

    Journal: PLoS ONE

    Article Title: Zfat -Deficiency Results in a Loss of CD3ζ Phosphorylation with Dysregulation of ERK and Egr Activities Leading to Impaired Positive Selection

    doi: 10.1371/journal.pone.0076254

    Figure Lengend Snippet: (A)Immunoblots for Egr1, Egr2 and Egr3 before or at the indicated time points after the stimulation with plate-bound anti-CD3ε and anti-CD28 antibodies in DP cells from the indicated genotypes. Actin was used as a loading control. Data are representative of three independent experiments. (B) Immunoblots for Egr1, Egr2 and Egr3 in DP cells from the indicated genotypes before or at the indicated time points after the stimulation with plate-bound anti-CD3ε and anti-CD28 antibodies under the condition with U0126 in DMSO or with DMSO alone. Actin was used as a loading control. Data are representative of three independent experiments. (C, D) Quantitative RT-PCR analysis of Egr1 , Egr2 and Egr3 expression before (C) or at the indicated time points after the stimulation with anti-CD3ε and anti-CD28 antibodies (D) in DP cells from Zfat f/f and Zfat f/f - LckCre mice. The relative expression for each gene was normalized by the expression of Actb . The results are presented as the value relative to the unstimulated DP cells from Zfat f/f mice. The data are the mean ± s.d.; n.s., not significant.

    Article Snippet: The antibodies used for immunoblotting analysis and their specificities were as follows: Egr3, phosphorylated ERK, antibodies for phosphorylated and total Zap70, PLCγ1, c-Raf, and Lck (all from Cell Signaling Technology); CREB1 (C-21), Erk (K-23) and Egr1 (C-19; all from Santa Cruz); total and phosphorylated MEK1/2 (from New England Biolabs); Actin (A2066; from Sigma); Egr2 (from Proteintech Group); CD3ζ (from Exbio); phosphorylated CD3ζ (A0468; from Assay biotech).

    Techniques: Western Blot, Quantitative RT-PCR, Expressing

    Patient characteristics.

    Journal: Scientific Reports

    Article Title: Expression and prognostic value of the transcription factors EGR1 and EGR3 in gliomas

    doi: 10.1038/s41598-020-66236-x

    Figure Lengend Snippet: Patient characteristics.

    Article Snippet: Incubation with primary EGR1 antibody 1:50 (clone: 15F7, Cell Signaling Technology) or EGR3 antibody 1:2000 (clone: PA5–40841, ThermoFisher Scientific) was done using the BenchMark ULTRA platform (Ventana Medical Systems) with the OptiView-DAB detection system for EGR1 stainings, and the AutostainerPlus platform (DAKO, Glostrup, Denmark) with the catalyzed signal amplification system II for EGR3 stainings.

    Techniques: Methylation

    EGR1 and EGR3 protein expression in gliomas. (A) EGR1 and EGR3 expression levels displayed in representative grade II, III and IV gliomas. Images were acquired at 40X magnification with scale bars = 50 μm. EGR1 protein expression significantly increased with WHO grade and the highest expression levels were found in glioblastomas. EGR3 protein expression levels were independent of WHO grade. ( B) Expression of both EGR1 and EGR3 was seen in areas with characteristic histological traits of GBMs, including areas with necrosis, pseudopalisades and around microvascular proliferations. Images were acquired at 30X magnification with scale bars = 100 μm. N = Necrosis. P = pseudo-palisade. M = microvascular proliferation. ( C) Verhaak subtype stratification of EGR1 mRNA TCGA data showed that neural and proneural glioblastomas have slightly lower EGR1 mRNA levels compared to classical and mesenchymal tumors, while EGR3 was equally expressed in all subtypes. ( D) Stratification of EGR1 and EGR3 protein levels based on IDH1/2 mutational status in the patient cohort. No significant changes in EGR1 or EGR3 protein levels were observed. * = P < 0.05. ** = P < 0.01. *** = P < 0.001.

    Journal: Scientific Reports

    Article Title: Expression and prognostic value of the transcription factors EGR1 and EGR3 in gliomas

    doi: 10.1038/s41598-020-66236-x

    Figure Lengend Snippet: EGR1 and EGR3 protein expression in gliomas. (A) EGR1 and EGR3 expression levels displayed in representative grade II, III and IV gliomas. Images were acquired at 40X magnification with scale bars = 50 μm. EGR1 protein expression significantly increased with WHO grade and the highest expression levels were found in glioblastomas. EGR3 protein expression levels were independent of WHO grade. ( B) Expression of both EGR1 and EGR3 was seen in areas with characteristic histological traits of GBMs, including areas with necrosis, pseudopalisades and around microvascular proliferations. Images were acquired at 30X magnification with scale bars = 100 μm. N = Necrosis. P = pseudo-palisade. M = microvascular proliferation. ( C) Verhaak subtype stratification of EGR1 mRNA TCGA data showed that neural and proneural glioblastomas have slightly lower EGR1 mRNA levels compared to classical and mesenchymal tumors, while EGR3 was equally expressed in all subtypes. ( D) Stratification of EGR1 and EGR3 protein levels based on IDH1/2 mutational status in the patient cohort. No significant changes in EGR1 or EGR3 protein levels were observed. * = P < 0.05. ** = P < 0.01. *** = P < 0.001.

    Article Snippet: Incubation with primary EGR1 antibody 1:50 (clone: 15F7, Cell Signaling Technology) or EGR3 antibody 1:2000 (clone: PA5–40841, ThermoFisher Scientific) was done using the BenchMark ULTRA platform (Ventana Medical Systems) with the OptiView-DAB detection system for EGR1 stainings, and the AutostainerPlus platform (DAKO, Glostrup, Denmark) with the catalyzed signal amplification system II for EGR3 stainings.

    Techniques: Expressing

    Expression of EGR1 and EGR3 in infiltrating tumour cells. (A–C) Representative HE, P53, and double-fluorescence images with P53/EGR1 on tissue sections from one of the tumours included in the cohort. 1 = central tumour area. 2 = intermediate tumour area. 3 = peripheral tumour area. Scale bar = 2.5 mm. DAPI-stained nuclei = blue, P53-positive nuclei = green, EGR1/3-positive nuclei = red, double-positive nuclei, i.e. positive for both P53 and EGR1/3 = orange. ( D,E) The EGR1 fraction and staining intensity in EGR1 + tumour cells was significantly reduced from central tumour to periphery. ( F) Ivy GAP data showed a trending but non-significant decline in EGR1 mRNA levels between central tumour and tumour periphery. ( G,H) The EGR3 fraction remained constant across the different tumour regions, while the mean staining intensity increased significantly in peripheral tumour cells compared to central tumour cells. I) Ivy Gap EGR3 mRNA data also showed an increase in EGR3 intensity in the intermediate and peripheral areas compared to central tumour. * = P < 0.05. ** = P < 0.01. *** = P < 0.001.

    Journal: Scientific Reports

    Article Title: Expression and prognostic value of the transcription factors EGR1 and EGR3 in gliomas

    doi: 10.1038/s41598-020-66236-x

    Figure Lengend Snippet: Expression of EGR1 and EGR3 in infiltrating tumour cells. (A–C) Representative HE, P53, and double-fluorescence images with P53/EGR1 on tissue sections from one of the tumours included in the cohort. 1 = central tumour area. 2 = intermediate tumour area. 3 = peripheral tumour area. Scale bar = 2.5 mm. DAPI-stained nuclei = blue, P53-positive nuclei = green, EGR1/3-positive nuclei = red, double-positive nuclei, i.e. positive for both P53 and EGR1/3 = orange. ( D,E) The EGR1 fraction and staining intensity in EGR1 + tumour cells was significantly reduced from central tumour to periphery. ( F) Ivy GAP data showed a trending but non-significant decline in EGR1 mRNA levels between central tumour and tumour periphery. ( G,H) The EGR3 fraction remained constant across the different tumour regions, while the mean staining intensity increased significantly in peripheral tumour cells compared to central tumour cells. I) Ivy Gap EGR3 mRNA data also showed an increase in EGR3 intensity in the intermediate and peripheral areas compared to central tumour. * = P < 0.05. ** = P < 0.01. *** = P < 0.001.

    Article Snippet: Incubation with primary EGR1 antibody 1:50 (clone: 15F7, Cell Signaling Technology) or EGR3 antibody 1:2000 (clone: PA5–40841, ThermoFisher Scientific) was done using the BenchMark ULTRA platform (Ventana Medical Systems) with the OptiView-DAB detection system for EGR1 stainings, and the AutostainerPlus platform (DAKO, Glostrup, Denmark) with the catalyzed signal amplification system II for EGR3 stainings.

    Techniques: Expressing, Fluorescence, Staining

    Overall patient survival in different subgroups. Patient survival illustrated for different EGR1 and EGR3 subgroups in glioblastoma patients both measured by positive cell fractions found in the immunostainings and by mRNA expression data from TCGA. All groups were dichotomized at the median. ( A) GBM patients with a high EGR1 nuclear fraction showed a significantly better prognosis compared to patients with a low EGR1 fraction. ( B) EGR1 mRNA levels from TCGA did not show any difference in survival between the two groups. ( C) Patients with a high EGR3 nuclear fraction were borderline significant of having a poor prognosis compared to patients with a low EGR3 fraction. ( D) EGR3 mRNA levels from TCGA showed the same trend as EGR3 protein levels. ( E) MGMT-gene promoter methylated patients with a high EGR3 fraction lived significantly shorter than patients with a low EGR3 fraction. ( F) The same trend was found in EGR3 mRNA data from TCGA, although results were non-significant . G) When sub-stratifying patients from the EGR1 high group by EGR3 expression, the group with high EGR3 expression had significantly shorter survival compared to patients with low EGR3 expression. H) Sub-stratification of patients from the EGR1 low group by EGR3 expression did not result in survival differences between the two groups. HR = Hazard ratio.

    Journal: Scientific Reports

    Article Title: Expression and prognostic value of the transcription factors EGR1 and EGR3 in gliomas

    doi: 10.1038/s41598-020-66236-x

    Figure Lengend Snippet: Overall patient survival in different subgroups. Patient survival illustrated for different EGR1 and EGR3 subgroups in glioblastoma patients both measured by positive cell fractions found in the immunostainings and by mRNA expression data from TCGA. All groups were dichotomized at the median. ( A) GBM patients with a high EGR1 nuclear fraction showed a significantly better prognosis compared to patients with a low EGR1 fraction. ( B) EGR1 mRNA levels from TCGA did not show any difference in survival between the two groups. ( C) Patients with a high EGR3 nuclear fraction were borderline significant of having a poor prognosis compared to patients with a low EGR3 fraction. ( D) EGR3 mRNA levels from TCGA showed the same trend as EGR3 protein levels. ( E) MGMT-gene promoter methylated patients with a high EGR3 fraction lived significantly shorter than patients with a low EGR3 fraction. ( F) The same trend was found in EGR3 mRNA data from TCGA, although results were non-significant . G) When sub-stratifying patients from the EGR1 high group by EGR3 expression, the group with high EGR3 expression had significantly shorter survival compared to patients with low EGR3 expression. H) Sub-stratification of patients from the EGR1 low group by EGR3 expression did not result in survival differences between the two groups. HR = Hazard ratio.

    Article Snippet: Incubation with primary EGR1 antibody 1:50 (clone: 15F7, Cell Signaling Technology) or EGR3 antibody 1:2000 (clone: PA5–40841, ThermoFisher Scientific) was done using the BenchMark ULTRA platform (Ventana Medical Systems) with the OptiView-DAB detection system for EGR1 stainings, and the AutostainerPlus platform (DAKO, Glostrup, Denmark) with the catalyzed signal amplification system II for EGR3 stainings.

    Techniques: Expressing, Methylation

    Multivariate Cox-regression for glioblastoma patients.

    Journal: Scientific Reports

    Article Title: Expression and prognostic value of the transcription factors EGR1 and EGR3 in gliomas

    doi: 10.1038/s41598-020-66236-x

    Figure Lengend Snippet: Multivariate Cox-regression for glioblastoma patients.

    Article Snippet: Incubation with primary EGR1 antibody 1:50 (clone: 15F7, Cell Signaling Technology) or EGR3 antibody 1:2000 (clone: PA5–40841, ThermoFisher Scientific) was done using the BenchMark ULTRA platform (Ventana Medical Systems) with the OptiView-DAB detection system for EGR1 stainings, and the AutostainerPlus platform (DAKO, Glostrup, Denmark) with the catalyzed signal amplification system II for EGR3 stainings.

    Techniques: Methylation, Mutagenesis

    MGMT-methylation distribution and multivariate cox-regression for EGR1/EGR3 combinations. (A) The distribution of MGMT-methylation status was highly skewed when looking at the two EGR1 groups: The majority of patients in the EGR1 high group had a methylated MGMT-promoter, while 100% of patients in the EGR1 low group had un-methylated MGMT-promoters. ( B) TCGA mRNA data showed a significant inverse correlation between EGR1 and MGMT mRNA levels. ( C) TCGA data showed that patients with recurrent GBM and high EGR3 mRNA levels had significantly shorter survival compared to patients with low EGR3 mRNA levels. ( D,E) Multivariate Cox-regression of the different EGR1 and EGR3 combinations showed that patients with EGR1 high/EGR3 high had a worse prognosis than patients with EGR1 high/EGR3 low. * = P < 0.05. ** = P < 0.01. *** = P < 0.001.

    Journal: Scientific Reports

    Article Title: Expression and prognostic value of the transcription factors EGR1 and EGR3 in gliomas

    doi: 10.1038/s41598-020-66236-x

    Figure Lengend Snippet: MGMT-methylation distribution and multivariate cox-regression for EGR1/EGR3 combinations. (A) The distribution of MGMT-methylation status was highly skewed when looking at the two EGR1 groups: The majority of patients in the EGR1 high group had a methylated MGMT-promoter, while 100% of patients in the EGR1 low group had un-methylated MGMT-promoters. ( B) TCGA mRNA data showed a significant inverse correlation between EGR1 and MGMT mRNA levels. ( C) TCGA data showed that patients with recurrent GBM and high EGR3 mRNA levels had significantly shorter survival compared to patients with low EGR3 mRNA levels. ( D,E) Multivariate Cox-regression of the different EGR1 and EGR3 combinations showed that patients with EGR1 high/EGR3 high had a worse prognosis than patients with EGR1 high/EGR3 low. * = P < 0.05. ** = P < 0.01. *** = P < 0.001.

    Article Snippet: Incubation with primary EGR1 antibody 1:50 (clone: 15F7, Cell Signaling Technology) or EGR3 antibody 1:2000 (clone: PA5–40841, ThermoFisher Scientific) was done using the BenchMark ULTRA platform (Ventana Medical Systems) with the OptiView-DAB detection system for EGR1 stainings, and the AutostainerPlus platform (DAKO, Glostrup, Denmark) with the catalyzed signal amplification system II for EGR3 stainings.

    Techniques: Methylation

    FBW7α targets SOX9 for ubiquitination and destruction after UV-induced DNA damage. ( A ) Screen of F-box proteins. H293T cells were co-transfected with FLAG-SOX9 and an empty vector or the GST-tagged F-box proteins FBXL3a, FBXL13, FBXL18, FBXO16, FBW4, FBW6 or FBW7, as well as green fluorescent protein (GFP) for a transfection control. Lysates were immunoblotted for FLAG and GST. α-Tubulin (α-TUB) was used as a loading control. ( B ) Western blot of FLAG-SOX9, HA-FBW7 and GFP of lysates from U2OS and H1299 cells that were co-transfected with FLAG-SOX9, GFP and increasing amounts of HA-FBW7α. α-tubulin was used as a loading control. ( C ) FBW7 mediates UV-induced SOX9 degradation. FBW7 +/+ or FBW 7 −/− HCT116 colon cancer cells UV irradiated (8 h, 80 J/m 2 ), or unirradiated, were immunoblotted for SOX9. GAPDH was used as a loading control. ( D ) FBW7 decreases endogenous SOX9. FBW7 −/− HCT116 were transfected with HA-FBW7. FBW7 +/+ and FBW7 −/− HCT116 cells were immunoblotted for SOX9 and HA-FBW7. GAPDH was used as a loading control. ( E ) RNA from FBW7 +/+ and FBW7 −/− HCT116 cells were examined for deletion of exon 5 in FBW7, utilizing primers that span exons 4 and 6 and that yield a 251 bp product. The absence of an RT-PCR product in lane 2 signifies deletion of exon 5 and de-stabilization of the mRNA. GAPDH was used as a control for mRNA quality.

    Journal: Nucleic Acids Research

    Article Title: SOX9 is targeted for proteasomal degradation by the E3 ligase FBW7 in response to DNA damage

    doi: 10.1093/nar/gkw748

    Figure Lengend Snippet: FBW7α targets SOX9 for ubiquitination and destruction after UV-induced DNA damage. ( A ) Screen of F-box proteins. H293T cells were co-transfected with FLAG-SOX9 and an empty vector or the GST-tagged F-box proteins FBXL3a, FBXL13, FBXL18, FBXO16, FBW4, FBW6 or FBW7, as well as green fluorescent protein (GFP) for a transfection control. Lysates were immunoblotted for FLAG and GST. α-Tubulin (α-TUB) was used as a loading control. ( B ) Western blot of FLAG-SOX9, HA-FBW7 and GFP of lysates from U2OS and H1299 cells that were co-transfected with FLAG-SOX9, GFP and increasing amounts of HA-FBW7α. α-tubulin was used as a loading control. ( C ) FBW7 mediates UV-induced SOX9 degradation. FBW7 +/+ or FBW 7 −/− HCT116 colon cancer cells UV irradiated (8 h, 80 J/m 2 ), or unirradiated, were immunoblotted for SOX9. GAPDH was used as a loading control. ( D ) FBW7 decreases endogenous SOX9. FBW7 −/− HCT116 were transfected with HA-FBW7. FBW7 +/+ and FBW7 −/− HCT116 cells were immunoblotted for SOX9 and HA-FBW7. GAPDH was used as a loading control. ( E ) RNA from FBW7 +/+ and FBW7 −/− HCT116 cells were examined for deletion of exon 5 in FBW7, utilizing primers that span exons 4 and 6 and that yield a 251 bp product. The absence of an RT-PCR product in lane 2 signifies deletion of exon 5 and de-stabilization of the mRNA. GAPDH was used as a control for mRNA quality.

    Article Snippet: Antibodies against the following proteins were purchased from Cell Signaling: GST (8146), FLAG (8146), HA (2367), GFP (2559), GSK3β (9315), Phospho-histone H3 (9701), Notch1-ICD (4147), phospho-CHK1 (2341), phospho-CHK2 (2661), phospho-ERK (9101), phospho-AKT (4060), phospho-CREB (9198), phospho-MAPKAPK2 (3007), PARP (9542), ATM (2873), ATR (2790), DNA-PK (4602) and GSK3β (9315).

    Techniques: Transfection, Plasmid Preparation, Western Blot, Irradiation, Reverse Transcription Polymerase Chain Reaction

    UV-induced SOX9 degradation requires GSK3 activity. ( A ) GSK3 enhances FBW7α-mediated SOX9 degradation. H1299 cells were co-transfected with HA-FBW7α and FLAG-SOX9, and were either untreated or treated with CHIR-99021 in the presence of CHX. Lysates were collected at varying times and probed for FLAG-SOX9 and HA-FBW7. The graph on the right depicts the half-life of SOX9, determined by densitometric quantitation. GAPDH was used as a normalization control. SE, short film exposure; LE, long film exposure. ( B ) H1299 cells were transfected with FLAG-SOX9 with or without wild-type (WT) GSK3β or constitutively active S9A GSK3β and with or without HA-FBW7α, which was transfected at a 1:4 molar ratio to the other plasmids. GFP was transfected as a control. Lysates were immunoblotted for FLAG-SOX9, HA-FBW7, GFP and GSK3β. Levels of FLAG-SOX9 were quantified by densitometry, normalized to GAPDH. ( C ) FBW7α interacts with SOX9. H1299 and U2OS cells were transfected with HA-FBW7α along with FLAG-SOX9. Lysates were immunoprecipitated with anti-FLAG and immunoblotted for FLAG-SOX9 and HA-FBW7. A Western blot of the 5% input lysates is also shown ( D ) Endogenous FBW7 interacts with endogenous SOX9. SOX9 was immunoprecipitated (IP) from H1299 lysates, and proteins were immunoblotted for FBW7 and SOX9, as well as IgG. A Western blot of the 5% input lysates is also shown. ( E ) Interaction between FBW7α and SOX9 is dependent on GSK3. H1299 cells were transfected with HA-FBW7α along with FLAG-SOX9 and GFP as a transfection control, either untreated or treated with the GSK3 inhibitor, CHIR99021. Lysates were immunoprecipitated with anti-FLAG and immunoblotted for FLAG-SOX9 and HA-FBW7. A Western blot of the 5% input lysates is also shown. ( F ) FBW7 targets SOX9 for ubiquitination. H1299 cells were co-transfected with either FLAG-SOX9 or FLAG-MUT-SOX9 (CPD-mutant), and HA-Ub with or without HA-FBW7α. Cells were treated with MG132 with or without CHIR99021 for 6 h before collection of lysates. GFP was transfected as a control. Denatured lysates were immunoprecipitated with anti-FLAG and immunoblotted for Ubiquitin. The 5% input lysates were also immunoblotted for HA, FLAG and GFP.

    Journal: Nucleic Acids Research

    Article Title: SOX9 is targeted for proteasomal degradation by the E3 ligase FBW7 in response to DNA damage

    doi: 10.1093/nar/gkw748

    Figure Lengend Snippet: UV-induced SOX9 degradation requires GSK3 activity. ( A ) GSK3 enhances FBW7α-mediated SOX9 degradation. H1299 cells were co-transfected with HA-FBW7α and FLAG-SOX9, and were either untreated or treated with CHIR-99021 in the presence of CHX. Lysates were collected at varying times and probed for FLAG-SOX9 and HA-FBW7. The graph on the right depicts the half-life of SOX9, determined by densitometric quantitation. GAPDH was used as a normalization control. SE, short film exposure; LE, long film exposure. ( B ) H1299 cells were transfected with FLAG-SOX9 with or without wild-type (WT) GSK3β or constitutively active S9A GSK3β and with or without HA-FBW7α, which was transfected at a 1:4 molar ratio to the other plasmids. GFP was transfected as a control. Lysates were immunoblotted for FLAG-SOX9, HA-FBW7, GFP and GSK3β. Levels of FLAG-SOX9 were quantified by densitometry, normalized to GAPDH. ( C ) FBW7α interacts with SOX9. H1299 and U2OS cells were transfected with HA-FBW7α along with FLAG-SOX9. Lysates were immunoprecipitated with anti-FLAG and immunoblotted for FLAG-SOX9 and HA-FBW7. A Western blot of the 5% input lysates is also shown ( D ) Endogenous FBW7 interacts with endogenous SOX9. SOX9 was immunoprecipitated (IP) from H1299 lysates, and proteins were immunoblotted for FBW7 and SOX9, as well as IgG. A Western blot of the 5% input lysates is also shown. ( E ) Interaction between FBW7α and SOX9 is dependent on GSK3. H1299 cells were transfected with HA-FBW7α along with FLAG-SOX9 and GFP as a transfection control, either untreated or treated with the GSK3 inhibitor, CHIR99021. Lysates were immunoprecipitated with anti-FLAG and immunoblotted for FLAG-SOX9 and HA-FBW7. A Western blot of the 5% input lysates is also shown. ( F ) FBW7 targets SOX9 for ubiquitination. H1299 cells were co-transfected with either FLAG-SOX9 or FLAG-MUT-SOX9 (CPD-mutant), and HA-Ub with or without HA-FBW7α. Cells were treated with MG132 with or without CHIR99021 for 6 h before collection of lysates. GFP was transfected as a control. Denatured lysates were immunoprecipitated with anti-FLAG and immunoblotted for Ubiquitin. The 5% input lysates were also immunoblotted for HA, FLAG and GFP.

    Article Snippet: Antibodies against the following proteins were purchased from Cell Signaling: GST (8146), FLAG (8146), HA (2367), GFP (2559), GSK3β (9315), Phospho-histone H3 (9701), Notch1-ICD (4147), phospho-CHK1 (2341), phospho-CHK2 (2661), phospho-ERK (9101), phospho-AKT (4060), phospho-CREB (9198), phospho-MAPKAPK2 (3007), PARP (9542), ATM (2873), ATR (2790), DNA-PK (4602) and GSK3β (9315).

    Techniques: Activity Assay, Transfection, Quantitation Assay, Immunoprecipitation, Western Blot, Mutagenesis

    Phosphorylation of SOX9 in the K2 domain by GSK3β triggers SOX9 destruction by FBW7α. ( A ) Alignment of the putative SOX9 consensus phosphodegron (CPD) sites (Motifs 1–4) with the FBW7 CPD sequence known for other FBW7 target proteins. The SOX9 serine and threonine residues that were mutated to alanine are underlined. ( B ) The FBW7α CPD binding site on SOX9 comprises amino acids 236–240. The top schematic depicts the locations of the DIM, HMG, K2, PQA and TA domains of SOX9, as well as the locations of the putative CPD motifs. Below, H1299 cells were transfected with WT FLAG-SOX9 or the indicated FLAG-SOX9 mutants (M1–M4) with GFP and with or without HA-FBW7α. Lysates were immunoblotted for GFP, FLAG and HA. GAPDH was used as a normalization control. SE, short film exposure; LE, long film exposure. ( C ) The SOX9 CPD sequence targeted by FBW7α is conserved across different species. The sequence shown for zebrafish is the SOX9a ortholog. ( D ) SOX9 interacts with FBW7α at the K2 domain CPD motif. H1299 cells were co-transfected with HA-FBW7α and either wild-type FLAG-SOX9 or CPD-mutant FLAG-MUT-SOX9. Lysates were immunoprecipitated with anti-FLAG and immunoblotted for HA and FLAG. A Western blot of lysates comprising 5% of input is also shown for HA and FLAG. ( E ) The SOX9 K2 domain is phosphorylated by GSK3β at T236. Beas2b cells were transfected with the FLAG-control, wild-type FLAG-SOX9 or CPD-mutant FLAG-MUT-SOX9. Cells were treated with 40 μM CHIR-99021 for 8 h, 48 h after transfection. A Western blot of lysates is shown for SOX9, P-T236-SOX9 and GAPDH. SE, short film exposure; LE, long film exposure. ( F ) FBW7α directly interacts with SOX9 in vitro . A binding assay was performed on HA-FBW7α protein and a GST-tagged WT SOX9 fragment comprising amino acids 186–318, or a GST-tagged SOX9 mutated at the CPD motif, with or without human recombinant GSK3β (rhGSK3β). The input and GST-immunoprecipitated proteins were immunoblotted for HA-FBW7. Total GST and GST-SOX9 were visualized by a coomassie blue stain. M, protein marker. ( G ) GSK3β directly phosphorylates SOX9. An in vitro kinase assay was performed using human recombinant GSK3β and purified GST, wild-type GST-SOX9 or a CPD-mutant GST-SOX9. 32 P GST-SOX9 is shown. Total GST and GST-SOX9 were visualized by a coomassie blue stain. Presence of GSK3β was confirmed by immunoblotting. Graph represents the average phosphorylation of CPD-mutant SOX relative to wild-type SOX9 in three independent experiments. Error bars represent the standard deviation. A two-tailed Student's t -test was used to compare groups. **, P < 0.01.

    Journal: Nucleic Acids Research

    Article Title: SOX9 is targeted for proteasomal degradation by the E3 ligase FBW7 in response to DNA damage

    doi: 10.1093/nar/gkw748

    Figure Lengend Snippet: Phosphorylation of SOX9 in the K2 domain by GSK3β triggers SOX9 destruction by FBW7α. ( A ) Alignment of the putative SOX9 consensus phosphodegron (CPD) sites (Motifs 1–4) with the FBW7 CPD sequence known for other FBW7 target proteins. The SOX9 serine and threonine residues that were mutated to alanine are underlined. ( B ) The FBW7α CPD binding site on SOX9 comprises amino acids 236–240. The top schematic depicts the locations of the DIM, HMG, K2, PQA and TA domains of SOX9, as well as the locations of the putative CPD motifs. Below, H1299 cells were transfected with WT FLAG-SOX9 or the indicated FLAG-SOX9 mutants (M1–M4) with GFP and with or without HA-FBW7α. Lysates were immunoblotted for GFP, FLAG and HA. GAPDH was used as a normalization control. SE, short film exposure; LE, long film exposure. ( C ) The SOX9 CPD sequence targeted by FBW7α is conserved across different species. The sequence shown for zebrafish is the SOX9a ortholog. ( D ) SOX9 interacts with FBW7α at the K2 domain CPD motif. H1299 cells were co-transfected with HA-FBW7α and either wild-type FLAG-SOX9 or CPD-mutant FLAG-MUT-SOX9. Lysates were immunoprecipitated with anti-FLAG and immunoblotted for HA and FLAG. A Western blot of lysates comprising 5% of input is also shown for HA and FLAG. ( E ) The SOX9 K2 domain is phosphorylated by GSK3β at T236. Beas2b cells were transfected with the FLAG-control, wild-type FLAG-SOX9 or CPD-mutant FLAG-MUT-SOX9. Cells were treated with 40 μM CHIR-99021 for 8 h, 48 h after transfection. A Western blot of lysates is shown for SOX9, P-T236-SOX9 and GAPDH. SE, short film exposure; LE, long film exposure. ( F ) FBW7α directly interacts with SOX9 in vitro . A binding assay was performed on HA-FBW7α protein and a GST-tagged WT SOX9 fragment comprising amino acids 186–318, or a GST-tagged SOX9 mutated at the CPD motif, with or without human recombinant GSK3β (rhGSK3β). The input and GST-immunoprecipitated proteins were immunoblotted for HA-FBW7. Total GST and GST-SOX9 were visualized by a coomassie blue stain. M, protein marker. ( G ) GSK3β directly phosphorylates SOX9. An in vitro kinase assay was performed using human recombinant GSK3β and purified GST, wild-type GST-SOX9 or a CPD-mutant GST-SOX9. 32 P GST-SOX9 is shown. Total GST and GST-SOX9 were visualized by a coomassie blue stain. Presence of GSK3β was confirmed by immunoblotting. Graph represents the average phosphorylation of CPD-mutant SOX relative to wild-type SOX9 in three independent experiments. Error bars represent the standard deviation. A two-tailed Student's t -test was used to compare groups. **, P < 0.01.

    Article Snippet: Antibodies against the following proteins were purchased from Cell Signaling: GST (8146), FLAG (8146), HA (2367), GFP (2559), GSK3β (9315), Phospho-histone H3 (9701), Notch1-ICD (4147), phospho-CHK1 (2341), phospho-CHK2 (2661), phospho-ERK (9101), phospho-AKT (4060), phospho-CREB (9198), phospho-MAPKAPK2 (3007), PARP (9542), ATM (2873), ATR (2790), DNA-PK (4602) and GSK3β (9315).

    Techniques: Sequencing, Binding Assay, Transfection, Mutagenesis, Immunoprecipitation, Western Blot, In Vitro, Recombinant, Staining, Marker, Kinase Assay, Purification, Standard Deviation, Two Tailed Test