anti phospho gsk3β ser21  (Bioss)

Journal: The Journal of Biological Chemistry

Article Title: Disruption of Ephx2 in cardiomyocytes but not endothelial cells improves functional recovery after ischemia-reperfusion in isolated mouse hearts

doi: 10.1016/j.jbc.2023.103049

Figure Lengend Snippet: Regulation of ERK and GSK3β phosphorylation during reperfusion after ischemia in Ephx2 fx/fx and Ephx2 fx/fx / Myh6-cre hearts treated with vehicle or EEZE. Detection and densitometric quantification of pERK and total ERK ( A and B ) or pGSK3β and total GSK3β ( C and D ) in Ephx2 fx/fx Cre negative and Ephx2 fx/fx /Myh6-cre heart lysates obtained under basal (nonischemic) conditions or after ischemia and 10 min of reperfusion, with or without ethanol vehicle or 1 μM EEZE pretreatment as indicated. N = 1 (non-ischemic) or N = 4 to 5 (I/R) as indicated, ∗ p < 0.05 versus Ephx2 fx/fx or vehicle. EEZE, 14,15-epoxyeicosa-5(Z)-enoic acid; EPHX, epoxide hydrolase; I/R, ischemia/reperfusion.

Article Snippet: Membranes were probed with antibodies to EPHX2 (sc-25797, 1:1000) and ERK1 (sc-93, 1:2000) from Santa Cruz Biotechnology, β-actin (AC-74, 1:5000) and pERK1/2 (M8159, 1:1000) from Sigma, and phospho-S21/9-GSK3β (9331) and GSK3β (9315) (1:1000 each) from Cell Signaling.

Techniques: Phospho-proteomics

Journal: Oncogenesis

Article Title: GSK3β palmitoylation mediated by ZDHHC4 promotes tumorigenicity of glioblastoma stem cells in temozolomide-resistant glioblastoma through the EZH2–STAT3 axis

doi: 10.1038/s41389-022-00402-w

Figure Lengend Snippet: A The collection of palmitoylated proteins and cancer driver genes in GBM. B The interaction of HA-ZDHHC4 with GFP-GSK3α and GFP-GSK3β was verified by immunoprecipitation in 293 T cells. C The localization of ZDHHC4 and GSK3β in SF126 cells was detected by immunofluorescence staining. D Protein map showing the three post-translationally modified amino acid residues in GSK3β. E SF126 cells were transfected, and the experiment was divided into three groups: wild-type Flag-GSK3β, C14A mutant GSK3β, and wild-type Flag-GSK3β were simultaneously knocked down ZDHHC4 by siRNA. ABE analysis and phosphorylation of GSK3α and GSK3β were performed in the three groups. The experiment was repeated twice. F ZDHHC4 was knocked down by siRNA in SF126 cells with stable expression of Flag-GSK3β. Immunoprecipitation assays showed the interaction of Flag-GSK3β with AKT, p70S6K, PKA, and p90RSK.

Article Snippet: Antibodies against GSK3β (12456), p- GSK3β (S9) (8566), GSK3α (4337), p- GSK3α (S21) (9316), EZH2 (5246), STAT3 (9139), p- STAT3 (Y705) (9145), β-catenin (8480), AKT1 (4691), and HA (3724) were purchased from Cell Signaling Technology.

Techniques: Immunoprecipitation, Immunofluorescence, Staining, Modification, Transfection, Mutagenesis, Phospho-proteomics, Expressing

Journal: Oncogenesis

Article Title: GSK3β palmitoylation mediated by ZDHHC4 promotes tumorigenicity of glioblastoma stem cells in temozolomide-resistant glioblastoma through the EZH2–STAT3 axis

doi: 10.1038/s41389-022-00402-w

Figure Lengend Snippet: A Statistical analysis of Figure A showed a positive correlation between p- STAT3 (Y705) level and ZDHHC4 expression. Data are shown as means ± SD ( n = 3). P -values were determined by two-tailed Student’s t -test. * P < 0.05; ** P < 0.01; *** P < 0.001. B Western blot analysis of the phosphorylation activity of GSK3β, EZH2, and STAT3 after treatment with ZDHHC4 siRNA. C Immunoprecipitation was used to detect the interaction between GSK3β, EZH2, and STAT3 in SF126 and U118 cells. D Schematic diagram of GSK3β regulating the EZH2–STAT3 axis.

Article Snippet: Antibodies against GSK3β (12456), p- GSK3β (S9) (8566), GSK3α (4337), p- GSK3α (S21) (9316), EZH2 (5246), STAT3 (9139), p- STAT3 (Y705) (9145), β-catenin (8480), AKT1 (4691), and HA (3724) were purchased from Cell Signaling Technology.

Techniques: Expressing, Two Tailed Test, Western Blot, Phospho-proteomics, Activity Assay, Immunoprecipitation

Journal: Oncogenesis

Article Title: GSK3β palmitoylation mediated by ZDHHC4 promotes tumorigenicity of glioblastoma stem cells in temozolomide-resistant glioblastoma through the EZH2–STAT3 axis

doi: 10.1038/s41389-022-00402-w

Figure Lengend Snippet: A Growth curve describing the effect of ZDHHC4 expression on TMZ inhibition in GBM cells. SF126 cells with overexpression or knockout of ZDHHC4 were treated with different concentrations of TMZ. B Representative image of colony formation in the SF126 cell lines 14 days after different treatments. For the control group, DMSO and TMZ were used. For the pLKO.1-ZDHHC4 group, DMSO, TMZ, and TMZ + Colivelin were used. For the pLVX-ZDHHC4 group, DMSO, TMZ, and TMZ + Stattic were used. Colonies were stained using crystal violet. C The number of clones stained by crystal violet in each dish in Figure B is calculated. Con-DMSO group was set at 100%. D ABE analysis of GSK3β palmitoylation in SF126 and SF126R cells. E Western blot analysis of GSK3β and STAT3 phosphorylation in SF126 and SF126R cells. F Immunoprecipitation analysis of the STAT3 binding capacity of GSK3β and EZH2 in SF126 and SF126R cells. G CCK-8 assay was used to detect the effect of GSK3β (C14A) mutant on TMZ killing SF126R cells. Data are shown as means ± SD ( n = 3). P -values were determined by two-tailed Student’s t -test. * P < 0.05; ** P < 0.01; *** P < 0.001.

Article Snippet: Antibodies against GSK3β (12456), p- GSK3β (S9) (8566), GSK3α (4337), p- GSK3α (S21) (9316), EZH2 (5246), STAT3 (9139), p- STAT3 (Y705) (9145), β-catenin (8480), AKT1 (4691), and HA (3724) were purchased from Cell Signaling Technology.

Techniques: Expressing, Inhibition, Over Expression, Knock-Out, Control, Staining, Clone Assay, Western Blot, Phospho-proteomics, Immunoprecipitation, Binding Assay, CCK-8 Assay, Mutagenesis, Two Tailed Test

Journal: Oncogenesis

Article Title: GSK3β palmitoylation mediated by ZDHHC4 promotes tumorigenicity of glioblastoma stem cells in temozolomide-resistant glioblastoma through the EZH2–STAT3 axis

doi: 10.1038/s41389-022-00402-w

Figure Lengend Snippet: A Representative images of GSCs induced from SF126R with ZDHHC4 knockdown. B Limiting dilution assay analysis (ELDA) of GSCs showed the frequencies of neurosphere formation. The significance of the difference between the indicated groups was determined by the χ 2 test ( n = 3 independent experiments). C Left, Real-time PCR analysis shows mRNA levels of STAT3 target stem-cell markers in ZDHHC4-knockdown SF126R GSCs. Control-shRNA cells were set to 1; right, Western blot verifies the effect of ZDHHC4 knockdown. D Left, SF126R cells with stable ZDHHC4 knockdown are supplemented with GSK3β or STAT3 for 24 h. Cell viability is measured by CCK-8. The cells stably expressing shNC transfected with empty vector were set to 1; Right, Expression verification of GSK3β and STAT3. Data are shown as means ± SD ( n = 3). P -values were determined by two-tailed Student’s t -test. * P < 0.05; ** P < 0.01; *** P < 0.001.

Article Snippet: Antibodies against GSK3β (12456), p- GSK3β (S9) (8566), GSK3α (4337), p- GSK3α (S21) (9316), EZH2 (5246), STAT3 (9139), p- STAT3 (Y705) (9145), β-catenin (8480), AKT1 (4691), and HA (3724) were purchased from Cell Signaling Technology.

Techniques: Knockdown, Limiting Dilution Assay, Real-time Polymerase Chain Reaction, Control, shRNA, Western Blot, CCK-8 Assay, Stable Transfection, Expressing, Transfection, Plasmid Preparation, Two Tailed Test

Journal: Oncogenesis

Article Title: GSK3β palmitoylation mediated by ZDHHC4 promotes tumorigenicity of glioblastoma stem cells in temozolomide-resistant glioblastoma through the EZH2–STAT3 axis

doi: 10.1038/s41389-022-00402-w

Figure Lengend Snippet: A U118R cells (shNC, shZDHHC4-1, and shZDHHC4-2, n = 5/group) (5 × 10 5 cells/mouse) were injected into nude mice. Mice were sacrificed 45 days later. H&E staining demonstrated typical tumor xenografts. B Intracranial tumor volumes in panel A were calculated (mean ± SD, n = 5 for each group, two-tailed Student’s t -test). C GSK3β palmitoylation and GSK3β-STAT3 pathway activity in tumors were detected by western blot. D The mRNA levels of GSC markers ( OCT4, NANOG, CD133, and SOX2 ) in tumor tissues at the end of the experiment were analyzed by RT-PCR. The folding changes were normalized to shNC (mean ± SD, n = 5 for each group, two-tailed Student’s t -test). E U118R cells (shNC and shZDHHC4-2 each in two groups, n = 5/group) were injected into nude mice. Three days after cell injection, mice were intraperitoneally injected with TMZ (25 mg kg −1 d −1 ) every other day for 30 days. Mice were sacrificed humanely 45 days later. H&E staining demonstrated typical tumor xenografts. F Intracranial tumor volumes in ( E ) were calculated (mean ± SD, n = 5 for each group, two-tailed Student’s t -test). G The mice were weighed every 4 days (mean ± SD, n = 5 for each group, two-tailed Student’s t -test). H Kaplan–Meier survival curves were used to define the overall survival of intracranial tumor-bearing mice.

Article Snippet: Antibodies against GSK3β (12456), p- GSK3β (S9) (8566), GSK3α (4337), p- GSK3α (S21) (9316), EZH2 (5246), STAT3 (9139), p- STAT3 (Y705) (9145), β-catenin (8480), AKT1 (4691), and HA (3724) were purchased from Cell Signaling Technology.

Techniques: Injection, Staining, Two Tailed Test, Activity Assay, Western Blot, Reverse Transcription Polymerase Chain Reaction

Journal: Oncogenesis

Article Title: GSK3β palmitoylation mediated by ZDHHC4 promotes tumorigenicity of glioblastoma stem cells in temozolomide-resistant glioblastoma through the EZH2–STAT3 axis

doi: 10.1038/s41389-022-00402-w

Figure Lengend Snippet: A Differences in ZDHHC4 expression in tumors and normal tissues of various organs obtained from the GENT database. B Relationship of ZDHHC4 to glioma grade from the REMBRANDT database. C , D Diagram of ZDHHC4 expression with overall survival and disease-free survival in glioma patients obtained from TCGA database. E Immunohistochemical staining showed that ZDHHC4 expression was correlated with p- GSK3β (Y216), p- STAT3 (Y705), and MGMT; 125 biologically independent samples were analyzed. The red dotted line marks the area corresponding to the high magnification image. F Statistical analysis of the Pearson correlation between the immunohistochemical staining score of ZDHHC4 expression and p- GSK3β (Y216) or p- STAT3 (Y705) expression.

Article Snippet: Antibodies against GSK3β (12456), p- GSK3β (S9) (8566), GSK3α (4337), p- GSK3α (S21) (9316), EZH2 (5246), STAT3 (9139), p- STAT3 (Y705) (9145), β-catenin (8480), AKT1 (4691), and HA (3724) were purchased from Cell Signaling Technology.

Techniques: Expressing, Immunohistochemical staining, Staining

Journal: Oncogenesis

Article Title: GSK3β palmitoylation mediated by ZDHHC4 promotes tumorigenicity of glioblastoma stem cells in temozolomide-resistant glioblastoma through the EZH2–STAT3 axis

doi: 10.1038/s41389-022-00402-w

Figure Lengend Snippet: ZDHHC4 palmitoylates GSK3β and competitively binds GSK3β with AKT and p70S6K kinases, which inhibits GSK3β Ser9 phosphorylation and enhances GSK3β Tyr216 phosphorylation. Activated GSK3β enhances GSC self-renewal, GBM TMZ-resistance, and tumorigenesis by promoting the EZH2/STAT3 axis.

Article Snippet: Antibodies against GSK3β (12456), p- GSK3β (S9) (8566), GSK3α (4337), p- GSK3α (S21) (9316), EZH2 (5246), STAT3 (9139), p- STAT3 (Y705) (9145), β-catenin (8480), AKT1 (4691), and HA (3724) were purchased from Cell Signaling Technology.

Techniques: Phospho-proteomics

Journal: bioRxiv

Article Title: Total parenteral nutrition drives glucose metabolism disorders by modulating gut microbiota and its metabolites

doi: 10.1101/2021.10.26.466009

Figure Lengend Snippet: Total parenteral nutrition impairs insulin sensitivity, liver glycogen deposition, insulin-dependent signaling and hepatic function in mice (n=8 each group, 3 times repeat). A-E. Intraperitoneal glucose tolerance test (A), intraperitoneal insulin tolerance test (B), fasting blood glucose level (C), fasting blood insulin level (D) and homeostasis model assessment of insulin resistance (HOMA-IR) value (E). * P<0.05, ** P<0.01, TPN group vs. Chow group. F-G. Representative histologic images showing deposition of glycogen in mouse hepatocytes (F) and quantification of the average levels of liver glycogen (G). H. Western blot (Left) and semiquantitative analyses (Right) of insulin-driven glycogen synthesis signaling (IRS1-Akt-GSK3) in the liver. I-J. Serum alanine transaminase (ALT) and aspartate transaminase (AST) concentrations.

Article Snippet: Antibodies against p-IRS1 (Ser307) (#2381), IRS1 (#2382), p-Akt (Ser473) (#4060), Akt (#9272), p-GSK3β (Ser9) (# 37F11), GSK3β (#5676), GAPDH (#2118) and β-actin (#4970) were used (1:1000 dilution; Cell Signaling Technology, Danvers, MA, USA).

Techniques: Western Blot

Journal: bioRxiv

Article Title: Total parenteral nutrition drives glucose metabolism disorders by modulating gut microbiota and its metabolites

doi: 10.1101/2021.10.26.466009

Figure Lengend Snippet: Total parenteral nutrition (TPN) alters the composition of the gut microbiota (n=5 each group, 3 times repeat). A. Principal coordinate analysis plot of Bray-Curtis distances. B. Bacterial diversity analysis based on operational taxonomic units (OTUs). C. Bacterial proportions at the phylum level. D. Bacterial discriminant analysis based on the linear discriminant analysis (LDA) score. E. Relative abundance of significantly different bacteria between the Chow and TPN groups. F. Schematic of the fecal microbiota transplantation experiments. G-H. Intraperitoneal glucose tolerance test (G) and intraperitoneal insulin tolerance test (H) in the Chow---+Abx and TPN---+Abx groups of mice.* P<0.05, ** P<0.01, TPN group vs. Chow group; # P<0.05, ## P<0.01, Chow---+Abx group vs TPN---+Abx group. I-J. Representative histologic images showing deposition of glycogen in mouse hepatocytes (I) and quantification of the average levels of liver glycogen (J). K. Western blot (Left) and semiquantitative analyses (Right) of insulin-driven glycogen synthesis signaling (IRS1-Akt-GSK3) in the liver. L. Lactobacillus abundance in patients with intestinal failure. L-PN: ≤80% of energy

Article Snippet: Antibodies against p-IRS1 (Ser307) (#2381), IRS1 (#2382), p-Akt (Ser473) (#4060), Akt (#9272), p-GSK3β (Ser9) (# 37F11), GSK3β (#5676), GAPDH (#2118) and β-actin (#4970) were used (1:1000 dilution; Cell Signaling Technology, Danvers, MA, USA).

Techniques: Bacteria, Transplantation Assay, Western Blot

Journal: bioRxiv

Article Title: Total parenteral nutrition drives glucose metabolism disorders by modulating gut microbiota and its metabolites

doi: 10.1101/2021.10.26.466009

Figure Lengend Snippet: Total parenteral nutrition (TPN) promotes poor insulin sensitivity by reducing the levels of tryptophan metabolites including indole-3-acetic acid (IAA) (n=5 each group, 3 times repeat). A. Principal component analysis based on metabolite composition. B. Volcano plot identifying differential serum metabolites between mice in the TPN and Chow groups. The outlying metabolites are indicated. C. Correlation analysis showing the associations between TPN-altered microbes and metabolites. D-E. Intraperitoneal glucose tolerance tests (D) and intraperitoneal insulin tolerance tests (E) for the four groups of mice.* P<0.05, ** P<0.01, TPN group vs. Chow group;# P<0.05, ## P<0.01, TPN+IAA group vs TPN+PBS group. F-G. Representative histologic images showing deposition of glycogen in mouse hepatocytes (F) and quantification of the average levels of liver glycogen (G). H. Western blot (Left) and semiquantitative analyses (Right) of insulin-driven glycogen synthesis signaling (IRSl-Akt-GSK3) in the liver. I. Serum concentration of lipopolysaccharide (LPS). J-K. Serum concentrations of IAA (J) and kynurenic acid (K) in patients with intestinal failure. L-PN: ≤80% of energy provided by PN; H-PN: >80% of energy provided by PN.

Article Snippet: Antibodies against p-IRS1 (Ser307) (#2381), IRS1 (#2382), p-Akt (Ser473) (#4060), Akt (#9272), p-GSK3β (Ser9) (# 37F11), GSK3β (#5676), GAPDH (#2118) and β-actin (#4970) were used (1:1000 dilution; Cell Signaling Technology, Danvers, MA, USA).

Techniques: Western Blot, Concentration Assay

Journal: bioRxiv

Article Title: Total parenteral nutrition drives glucose metabolism disorders by modulating gut microbiota and its metabolites

doi: 10.1101/2021.10.26.466009

Figure Lengend Snippet: Inactivation of indole/aryl hydrocarbon receptor signaling contributes to parenteral nutrition-related impairment of insulin sensitivity. A-B. Intraperitoneal glucose tolerance tests (A) and intraperitoneal insulin tolerance tests (B).(* P<0.05, ** P<0.01, Chow+DMSO group vs. Chow+CH223191 group;# P<0.05, ## P<0.01, Chow+CH223191 group vs Chow+CH22319+IAA group.) C-D. Representative histologic images showing deposition of glycogen in mouse hepatocytes (C) and quantification of the average levels of liver glycogen (D) E. Western blot (Left) and semiquantitative analyses (Right) of insulin-driven glycogen synthesis signaling (IRS1-Akt-GSK3) in mouse liver. (n=5 each group, 3 times repeat). F-G. Intraperitoneal glucose tolerance tests (F) and intraperitoneal insulin tolerance tests (G). *P<0.05, ** P<0.01, Chow+DMSO group vs. Chow+CH223191 group;# P<0.05, ## P<0.01, TPN+DMSO group vs TPN+Ficz group. H-I. Representative histologic images showing deposition of glycogen in mouse hepatocytes (H) and quantification of the average levels of liver glycogen (I). J. Western blot (Left) and semiquantitative ana1 ses (Right) of insulin-driven glycogen synthesis signaling (IRS1-Akt-GSK3) in the liver (n=8 each group, 3 times repeat).

Article Snippet: Antibodies against p-IRS1 (Ser307) (#2381), IRS1 (#2382), p-Akt (Ser473) (#4060), Akt (#9272), p-GSK3β (Ser9) (# 37F11), GSK3β (#5676), GAPDH (#2118) and β-actin (#4970) were used (1:1000 dilution; Cell Signaling Technology, Danvers, MA, USA).

Techniques: Western Blot

Journal: bioRxiv

Article Title: Total parenteral nutrition drives glucose metabolism disorders by modulating gut microbiota and its metabolites

doi: 10.1101/2021.10.26.466009

Figure Lengend Snippet: Inactivation of indole/aryl hydrocarbon receptor signaling reduces glucagon-like peptide-I production to cause parenteral nutrition (PN)-related impairment of insulin sensitivity. (n=8 each group, 3 times repeat) A. Serum glucagon-like peptide-I (GLP-I) concentration in patients with intestinal failure. L-PN: ≤80% of energy provided by PN; H-PN: >80% of energy provided by PN. B. Serum GLP-I concentration in the four groups of mice. C. Immunostaining of GLP-I in mouse terminal ileum and colon. D-E. Intraperitoneal glucose tolerance tests (D) and intraperitoneal insulin tolerance tests (E). * P<0.05, ** P<0.01, Chow+NS group vs. TPN+NS group;# P<0.05, ## P<0.01, TPN+NS group vs TPN+Liraglutide group. F-G. Representative histologic images showing deposition of glycogen in mouse hepatocytes (F) and quantification of the average levels of liver glycogen (G). H. Western blot (Left) and semiquantitative analyses (Right) of insulin-driven glycogen synthesis signaling (IRSI-Akt-GSK3) in the liver. I. Serum lipopolysaccharide (LPS) concentration.

Article Snippet: Antibodies against p-IRS1 (Ser307) (#2381), IRS1 (#2382), p-Akt (Ser473) (#4060), Akt (#9272), p-GSK3β (Ser9) (# 37F11), GSK3β (#5676), GAPDH (#2118) and β-actin (#4970) were used (1:1000 dilution; Cell Signaling Technology, Danvers, MA, USA).

Techniques: Concentration Assay, Immunostaining, Western Blot