rabbit  (Boster Bio)

Journal: Acta Pharmaceutica Sinica. B

Article Title: Reversing metabolic reprogramming by CPT1 inhibition with etomoxir promotes cardiomyocyte proliferation and heart regeneration via DUSP1 ADP-ribosylation-mediated p38 MAPK phosphorylation

doi: 10.1016/j.apsb.2024.11.001

Figure Lengend Snippet: DUPS1 regulates p38 MAPK signaling after CPT1 inhibition in postnatal cardiomyocytes. (A) The expression of Map2k3 , mitogen-activated protein kinase kinase 6 ( Map2k6 ), and dual specificity phosphatase family ( Dusp1 , Dusp2 , Dusp3 , Dusp4 , Dusp5 , Dusp6 , Dusp7 , Dusp8 , Dusp9 , Dusp10 , Dusp11 , Dusp12 , Dusp13 , Dusp14 , Dusp15 , Dusp16 , Dusp18 , Dusp19 , Dusp21 , Dusp22 ) in P1 and P7 cardiomyocytes ( n = 6, ∗ P < 0.05 vs. P1). (B) The effect of ETX treatment and Dusp1 siRNA (si Dusp1 ) on DUSP1 expression and p38 MAPK phosphorylation in P7 cardiomyocytes ( n = 3, ∗ P < 0.05 vs. Control, # P < 0.05 vs. ETX). (C) The effect of ETX treatment on the interaction of DUSP1 and p38 MAPK in P7 cardiomyocytes ( n = 6, ∗ P < 0.05 vs. Control). Error bars indicate SEM.

Article Snippet: The primary antibodies were: anti-phospho p38 mitogen-activated protein kinase (p-p38 MAPK) alpha Thr180/Tyr182 antibody (Thermo Fisher Scientific; 36-8500, Rabbit, 1:500), anti-p38 MAPK antibody (Cell Signaling Technology; 9212, Rabbit, 1:1000), anti-dual-specificity phosphatases 1 (DUSP1) antibody (Cell Signaling Technology; 48625, Rabbit, 1:1000), anti-dual-specificity phosphatases 4 (DUSP4) antibody (Cell Signaling Technology; 5149, Rabbit, 1:1000), anti-dual-specificity phosphatases 12 (DUSP12) antibody (Proteintech, Wuhan, China; 67101-1-Ig, Mouse, 1:1000), anti-Poly/Mono-ADP ribose antibody (Cell Signaling Technology; 83732, Rabbit, 1:1000), anti-poly(ADP-ribose) polymerase family, member 1 (PARP1) antibody (Proteintech; 66520-1-Ig, Mouse, 1:1000), anti-mitogen-activated protein kinase 3 (MAP2K3) antibody (Proteintech; 80137-1-RR, Rabbit, 1:1000), anti-CPT1A antibody (Abcam; ab234111, Rabbit, 1:1000), anti-CPT1B antibody (Proteintech; 22170-1-AP, Rabbit, 1:1000), and anti-glucokinase (GCK) antibody (Proteintech; 19666-1-AP, Rabbit, 1:1000).

Techniques: Inhibition, Expressing, Phospho-proteomics, Control

Journal: Translational Oncology

Article Title: DUSP4 enhances therapeutic sensitivity in HER2-positive breast cancer by inhibiting the G6PD pathway and ROS metabolism by interacting with ALDOB

doi: 10.1016/j.tranon.2024.102016

Figure Lengend Snippet: DUSP4 expression is higher in HER2-positive breast cancer patients who achieved pCR after neoadjuvant therapy. (A) Expression levels of DUSP4 in breast cancer patients who received PCH-based neoadjuvant therapy ( n = 86, 41 pCR vs. 45 non-pCR) were quantitated by real-time qPCR. (B) DUSP4 expression level in the lapatinib-resistant group and lapatinib-sensitive group, data from E-MEXP-440 dataset. DUSP4 expression level in the trastuzumab-resistant group and trastuzumab-sensitive group, data from GSE69017 dataset. (C) Relative DUSP4 expression in baseline and treated organoids. Normalized to ACTB. (D) DUSP4-overexpressing and pCDH control SKBR3 cells were analyzed by immunoblotting. DUSP4-knockout BT474 cells generated with CRISPR/Cas9 and control cells were analyzed by immunoblotting. Normalized to ACTB. (E) IC 50 values of trastuzumab, docetaxel and lapatinib in SKBR3 cells. (F) IC 50 values of trastuzumab, docetaxel and lapatinib in BT474 cells.

Article Snippet: Then, the sections were incubated with primary antibody (anti-DUSP4, CST, 1:100) overnight at 4 °C, incubated with goat anti-rabbit IgG and streptavidin peroxidase (SP) complex at room temperature for 30 min, and stained with DAB reagent (Gene Tech).

Techniques: Expressing, Western Blot, Knock-Out, Generated, CRISPR

Journal: Translational Oncology

Article Title: DUSP4 enhances therapeutic sensitivity in HER2-positive breast cancer by inhibiting the G6PD pathway and ROS metabolism by interacting with ALDOB

doi: 10.1016/j.tranon.2024.102016

Figure Lengend Snippet: DUSP4 enhances the sensitivity of HER2-positive breast cancer cells to combined treatment. (A) The drug combination effect of docetaxel and trastuzumab or docetaxel and lapatinib in cells with DUSP4 KO and NC. The color depth of the heatmap indicates the cell activity detected by CCK-8 assay. (B) The drug combination effect of docetaxel and trastuzumab or docetaxel and lapatinib in cells with DUSP4 overexpression and control. The color depth of the heatmap indicates the cell activity detected by CCK-8 assay.

Article Snippet: Then, the sections were incubated with primary antibody (anti-DUSP4, CST, 1:100) overnight at 4 °C, incubated with goat anti-rabbit IgG and streptavidin peroxidase (SP) complex at room temperature for 30 min, and stained with DAB reagent (Gene Tech).

Techniques: Activity Assay, CCK-8 Assay, Over Expression

Journal: Translational Oncology

Article Title: DUSP4 enhances therapeutic sensitivity in HER2-positive breast cancer by inhibiting the G6PD pathway and ROS metabolism by interacting with ALDOB

doi: 10.1016/j.tranon.2024.102016

Figure Lengend Snippet: DUSP4 immunohistochemical levels were positively associated with the prognosis of HER2-positive breast cancer. (A) DUSP4 expression in normal breast tissue and cancer tissue. (B) The expression of DUSP4 in different molecular types of breast cancer. (C) Correlation between DUSP4 and ERBB2 in breast cancer tissue. All the above were analyzed from TCGA data. * p < 0.05. (D) DUSP4 expression was determined by IHC staining in the TMA of HER2-positive breast cancer patients. Immunohistochemical photomicrographs are magnified 10X and 40X. The scale stands for 100 μm. (E) Relationship between DUSP4 expression and DFS/OS. P values were calculated using the unadjusted log-rank test.

Article Snippet: Then, the sections were incubated with primary antibody (anti-DUSP4, CST, 1:100) overnight at 4 °C, incubated with goat anti-rabbit IgG and streptavidin peroxidase (SP) complex at room temperature for 30 min, and stained with DAB reagent (Gene Tech).

Techniques: Immunohistochemical staining, Expressing, Immunohistochemistry

Journal: Translational Oncology

Article Title: DUSP4 enhances therapeutic sensitivity in HER2-positive breast cancer by inhibiting the G6PD pathway and ROS metabolism by interacting with ALDOB

doi: 10.1016/j.tranon.2024.102016

Figure Lengend Snippet: Multivariate regression analysis of DFS in patients with HER2-positive BC.

Article Snippet: Then, the sections were incubated with primary antibody (anti-DUSP4, CST, 1:100) overnight at 4 °C, incubated with goat anti-rabbit IgG and streptavidin peroxidase (SP) complex at room temperature for 30 min, and stained with DAB reagent (Gene Tech).

Techniques:

Journal: Translational Oncology

Article Title: DUSP4 enhances therapeutic sensitivity in HER2-positive breast cancer by inhibiting the G6PD pathway and ROS metabolism by interacting with ALDOB

doi: 10.1016/j.tranon.2024.102016

Figure Lengend Snippet: Loss of DUSP4 causes activation of the reactive oxygen species (ROS)-associated signaling pathway and reactive oxygen species removal. (A) GSEA showed the indicated pathway signatures in the DUSP4 KO vs. NC group in BT474 cells. (B) Significantly enriched genes in the ROS and NRF2 signaling pathways were coenriched with 6 genes including G6PD. (C) The expression levels of 6 genes were quantitated by real-time qPCR in DUSP4 KO vs. NC group in BT474 cells. (D) The expression levels of 6 genes were quantitated by real-time qPCR in DUSP4-overexpressing vs. pCDH group in SKBR3 cells. (E) ROS levels were detected by flow cytometry in DUSP4-overexpressing and control cells. (F) ROS levels were detected by flow cytometry in DUSP4-KO cells and NC cells. (G) ROS levels in the above cells were calculated and presented in a column diagram. (H) The pH value of the cell culture medium was detected at the indicated time points. (I) Cell viability testing with pyrotinib and NAC combination in BT474 cells. (J) ​ROS levels in BT474 cells in combination with pyrotinib and NAC. * p < 0.05.

Article Snippet: Then, the sections were incubated with primary antibody (anti-DUSP4, CST, 1:100) overnight at 4 °C, incubated with goat anti-rabbit IgG and streptavidin peroxidase (SP) complex at room temperature for 30 min, and stained with DAB reagent (Gene Tech).

Techniques: Activation Assay, Expressing, Flow Cytometry, Cell Culture

Journal: Translational Oncology

Article Title: DUSP4 enhances therapeutic sensitivity in HER2-positive breast cancer by inhibiting the G6PD pathway and ROS metabolism by interacting with ALDOB

doi: 10.1016/j.tranon.2024.102016

Figure Lengend Snippet: DUSP4 interacts with ALDOB. (A) Silver staining of immunoprecipitated proteins with DUSP4 antibody followed by SDS‒PAGE and subjected to MS analysis. (B) The top ten DUSP4-interacting proteins according to the score of identified unique peptides. (C) Immunoprecipitation of proteins with DUSP4 antibody was immunoblotted with ALDOB antibody. (C) Immunoprecipitation of proteins with ALDOB antibody was immunoblotted with DUSP4 antibody.

Article Snippet: Then, the sections were incubated with primary antibody (anti-DUSP4, CST, 1:100) overnight at 4 °C, incubated with goat anti-rabbit IgG and streptavidin peroxidase (SP) complex at room temperature for 30 min, and stained with DAB reagent (Gene Tech).

Techniques: Silver Staining, Immunoprecipitation

Journal: Translational Oncology

Article Title: DUSP4 enhances therapeutic sensitivity in HER2-positive breast cancer by inhibiting the G6PD pathway and ROS metabolism by interacting with ALDOB

doi: 10.1016/j.tranon.2024.102016

Figure Lengend Snippet: DUSP4 regulates G6PD activity through dephosphorylation of ALDOB . (A) ALDOB and G6PD were immunoblotted in DUSP4 stable BT474 cells. (B) A three-dimensional model of ALDOB protein was constructed. Cyan spheres represent positively charged amino acids, and red spheres represent negatively charged amino acids. The upper box: the specific area with 2 key points responsible for the interaction with G6PD. The lower box: Blue spheres represent the phosphorylated amino acid sites, while T39, T124 and T123 are threonine phosphorylation sites close to the above key sites. (C) Proteins immunoprecipitated with ALDOB antibody in SKBR3 cells transfected with wild-type or DUSP4 were immunoblotted as shown. (D) Proteins immunoprecipitated with ALDOB antibody in SKBR3 cells transfected with wild-type or mutated ALDOB were immunoblotted as shown. (E) G6PD activity was detected in SKBR3 DUSP4 stable cells and BT474 DUSP4 KO cells that were separately transfected with ALDOB wild-type plasmids, ALDOB T39A mutant plasmids or T123/124A plasmids. (F) The expression of G6PD in different molecular types of breast cancer. (G) The correlation between G6PD expression and relapse-free survival (RFS) was analyzed with Kaplan–Meier curves in HER2-positive breast cancer patients. All the above were analyzed based on TCGA data. * P < 0.05. (H) Schematic diagram of this study. DUSP4 dephosphorylates ALDOB and then augments the ALDOB/G6PD complex to inhibit G6PD activity and activate ROS signaling, ultimately increasing HER2 therapy sensitivity.

Article Snippet: Then, the sections were incubated with primary antibody (anti-DUSP4, CST, 1:100) overnight at 4 °C, incubated with goat anti-rabbit IgG and streptavidin peroxidase (SP) complex at room temperature for 30 min, and stained with DAB reagent (Gene Tech).

Techniques: Activity Assay, De-Phosphorylation Assay, Construct, Immunoprecipitation, Transfection, Mutagenesis, Expressing

Journal: Frontiers in Aging Neuroscience

Article Title: O-GlcNAcylation regulates extracellular signal-regulated kinase (ERK) activation in Alzheimer’s disease

doi: 10.3389/fnagi.2023.1155630

Figure Lengend Snippet: Extracellular signal-regulated kinase (ERK) signaling cascade in AD: activation of receptor tyrosine kinase (RTK) or APOE receptor (APOER) leads to the activation of Ras/Raf protein which in turn phosphorylates and activates MEK. MEK phosphorylates and activates ERK. Once ERK is activated it activates numerous downstream targets both in the cytosol and nucleus. Activation of nuclear transcription factors by ERK leads to increased transcription of APP gene and hence increased APP protein production. Increased level of APP protein potentially leads to increased Aβ plaque formation. ERK is inactivated by de-phosphorylation via dual specificity phosphatases such as DUSP4.

Article Snippet: Antibodies used in these studies were as follows: O -GlcNAc (RL2, ThermoFisher #MA1-072), actin (Sigma Catalog # A5316), α-Tubulin (sigma Catalog # T5168), OGT (AL-34) and OGA (345) antibodies were a generous gift from the laboratory of Dr. Gerald Hart (Department of Biological Chemistry, University of Georgia), Phospho-ERK (CST Catalog #9101), Total-ERK (Santa Cruz, Catalog # sc-93), phospho-MEK (ThermoFisher Catalog # 44-454G), Total MEK (ThermoFisher Catalog # 13-3500), APP (CST Catalog # 2452), and DUSP4 (CST Catalog # 5149).

Techniques: Activation Assay, De-Phosphorylation Assay

Journal: Frontiers in Aging Neuroscience

Article Title: O-GlcNAcylation regulates extracellular signal-regulated kinase (ERK) activation in Alzheimer’s disease

doi: 10.3389/fnagi.2023.1155630

Figure Lengend Snippet: Western blot analysis of samples harvested after serum reactivation time course of SH-SY5Y cells treated with (A) TMG as in , or subjected to panel (D) OGT KD 605, (G) OGT KD 606, (J) OGA KD 040, and (M) OGA KD 877. The blots were probed for p-MEK 1/2, MEK 1/2, Dusp4 and α-Tubulin. (B,E,H,K,N) Densitometry plot of MEK 1/2 phosphorylation normalized to total MEK 1/2 in SH-SY5Y cells with (B) TMG ( n = 3), (E) OGT KD 605 ( n = 4), (H) OGT KD 606 ( n = 4), (K) OGA KD 040 ( n = 3), and (N) OGA KD 877 ( n = 3). Densitometry plot of DUSP4 normalized to α-tubulin in SH-SY5Y cells with (C) TMG ( n = 3) (F) OGT KD 605 ( n = 4) (I) OGT KD 606 ( n = 4) (L) OGA KD 040 ( n = 3), and (O) OGA KD 877 ( n = 3), where n = total number of experimental trials. The dots represent the number of experimental trials (n). Statistical significance was measured using paired- t -test analysis and p -value is indicated on the plots. *Indicates p -values that are significant ( p < 0.05).

Article Snippet: Antibodies used in these studies were as follows: O -GlcNAc (RL2, ThermoFisher #MA1-072), actin (Sigma Catalog # A5316), α-Tubulin (sigma Catalog # T5168), OGT (AL-34) and OGA (345) antibodies were a generous gift from the laboratory of Dr. Gerald Hart (Department of Biological Chemistry, University of Georgia), Phospho-ERK (CST Catalog #9101), Total-ERK (Santa Cruz, Catalog # sc-93), phospho-MEK (ThermoFisher Catalog # 44-454G), Total MEK (ThermoFisher Catalog # 13-3500), APP (CST Catalog # 2452), and DUSP4 (CST Catalog # 5149).

Techniques: Western Blot, Phospho-proteomics

Journal: Frontiers in Aging Neuroscience

Article Title: O-GlcNAcylation regulates extracellular signal-regulated kinase (ERK) activation in Alzheimer’s disease

doi: 10.3389/fnagi.2023.1155630

Figure Lengend Snippet: (A–D) Western blot analysis of total brain lysate of WT-C57BL/6J male mice subjected to intra-peritoneal TMG injection for 1 month. The blots were probed for panel (A) O-GlcNAc, OGT, OGA, α-Tubulin (B) p-ERK 1/2, Total ERK 1, and APP. (C) Densitometry plot of ERK 1/2 phosphorylation normalized to total ERK 1 and (D) APP normalized to α-Tubulin. The dots represent the number of mice ( n = 4 for control and n = 5 for TMG injected mice). Statistical significance was measured using un-paired- t -test analysis and p -value is indicated on the plots. (E–K) Western blot analysis of total brain lysate of WT-C57BL/6J male mice subjected to intra-peritoneal TMG injection for 6 months. The blots were probed for panel (E) O-GlcNAc, OGT, OGA, α-Tubulin (F) p-ERK 1/2, Total ERK 1, and APP (I) p-MEK 1/2, MEK 1/2, Dusp4 and α-Tubulin. Densitometry plot of panel (G) ERK 1/2 phosphorylation normalized to total ERK 1 (H) APP normalized to α-Tubulin. (J) MEK 1/2 phosphorylation normalized to total MEK 1/2 and (K) DUSP4 normalized to α-tubulin. The dots represent the number of mice ( n = 4 for control and n = 5 for TMG injected mice). Statistical significance was measured using un-paired- t -test analysis and p -value is indicated on the plots. (L,M) Western blot analysis of SH-SY5Y neuroblastoma cells subjected to long term OGA inhibition with TMG treatment. The blots were probed for panel (L) O-GlcNAc, OGT, OGA, APP, α-Tubulin. (M) Densitometry plot of APP normalized to α-Tubulin. The dots represent the number of the number of experimental trials (n). Statistical significance was measured using paired- t -test analysis and p -value is indicated on the plots. **Indicates p -values that are significant and less than 0.01 ( p < 0.01).

Article Snippet: Antibodies used in these studies were as follows: O -GlcNAc (RL2, ThermoFisher #MA1-072), actin (Sigma Catalog # A5316), α-Tubulin (sigma Catalog # T5168), OGT (AL-34) and OGA (345) antibodies were a generous gift from the laboratory of Dr. Gerald Hart (Department of Biological Chemistry, University of Georgia), Phospho-ERK (CST Catalog #9101), Total-ERK (Santa Cruz, Catalog # sc-93), phospho-MEK (ThermoFisher Catalog # 44-454G), Total MEK (ThermoFisher Catalog # 13-3500), APP (CST Catalog # 2452), and DUSP4 (CST Catalog # 5149).

Techniques: Western Blot, Injection, Phospho-proteomics, Control, Inhibition

Journal: Frontiers in Aging Neuroscience

Article Title: O-GlcNAcylation regulates extracellular signal-regulated kinase (ERK) activation in Alzheimer’s disease

doi: 10.3389/fnagi.2023.1155630

Figure Lengend Snippet: Western blot analysis of total brain lysate of 5XFAD mice subjected to intra-peritoneal TMG injection for 1 month. The blots were probed for panel (A) O-GlcNAc, OGA, OGT, α-Tubulin (B) p-ERK 1/2, Total ERK 1, and APP (D) p-MEK 1/2, MEK 1/2, Dusp4 and α-Tubulin. Densitometry plot of panel (C) ERK 1/2 phosphorylation normalized to total ERK 1 (E) MEK 1/2 phosphorylation normalized to total MEK 1/2 and (F) DUSP4 normalized to α-tubulin in 5XFAD mice brains. The dots represent the number of mice ( n = 6 for control and n = 7 for TMG injected mice). Statistical significance was measured using un-paired- t -test analysis and p -value is indicated on the plots. *Indicates p -values that are significant ( p < 0.05).

Article Snippet: Antibodies used in these studies were as follows: O -GlcNAc (RL2, ThermoFisher #MA1-072), actin (Sigma Catalog # A5316), α-Tubulin (sigma Catalog # T5168), OGT (AL-34) and OGA (345) antibodies were a generous gift from the laboratory of Dr. Gerald Hart (Department of Biological Chemistry, University of Georgia), Phospho-ERK (CST Catalog #9101), Total-ERK (Santa Cruz, Catalog # sc-93), phospho-MEK (ThermoFisher Catalog # 44-454G), Total MEK (ThermoFisher Catalog # 13-3500), APP (CST Catalog # 2452), and DUSP4 (CST Catalog # 5149).

Techniques: Western Blot, Injection, Phospho-proteomics, Control

Journal: Frontiers in Aging Neuroscience

Article Title: O-GlcNAcylation regulates extracellular signal-regulated kinase (ERK) activation in Alzheimer’s disease

doi: 10.3389/fnagi.2023.1155630

Figure Lengend Snippet: Changes in O-GlcNAcylation amplify ERK 1/2 signaling. Arrows in magenta indicate long term OGA inhibition via TMG treatment for at least 2 weeks. Green arrows indicate OGT KD via shRNA 605. Blue arrows indicate OGT KD via shRNA 606. Purple arrows indicate OGA KD via shRNA 040. Orange arrows indicate OGA KD via shRNA 877. OGA inhibition via long term TMG treatment, OGT KD and OGA KD, all increase the amplitude of ERK 1/2 phosphorylation. TMG treatment increases ERK 1/2 phosphorylation by increasing the amplitude of MEK 1/2 phosphorylation. The levels of DUSP4 does not change with TMG treatment. OGT KD 606 also increases ERK 1/2 phosphorylation by increasing the amplitude of MEK 1/2 phosphorylation. The levels of DUSP4 decrease with OGT KD 606. OGT KD with shRNA 605 follows a similar trend as OGT KD with shRNA 606 where the levels of DUSP4 decreases. But the increase in MEK 1/2 phosphorylation by OGT KD 605 is not significant. OGA KD with both shRNA 040 and 877 increase ERK 1/2 phosphorylation but increase in the level of MEK 1/2 phosphorylation was not significant. DUSP4 decreases significantly with OGA KD 877 and DUSP4 levels do not change with OGA KD 040. Long term OGA inhibition also increased APP protein levels, correlating with increased ERK phosphorylation.

Article Snippet: Antibodies used in these studies were as follows: O -GlcNAc (RL2, ThermoFisher #MA1-072), actin (Sigma Catalog # A5316), α-Tubulin (sigma Catalog # T5168), OGT (AL-34) and OGA (345) antibodies were a generous gift from the laboratory of Dr. Gerald Hart (Department of Biological Chemistry, University of Georgia), Phospho-ERK (CST Catalog #9101), Total-ERK (Santa Cruz, Catalog # sc-93), phospho-MEK (ThermoFisher Catalog # 44-454G), Total MEK (ThermoFisher Catalog # 13-3500), APP (CST Catalog # 2452), and DUSP4 (CST Catalog # 5149).

Techniques: Inhibition, shRNA, Phospho-proteomics