p tyr216 gsk3β  (Bio-Rad)

Journal: Frontiers in Pharmacology

Article Title: hERG activators exhibit antitumor effects in breast cancer through calcineurin and β-catenin-mediated signaling pathways

doi: 10.3389/fphar.2025.1545300

Figure Lengend Snippet: Mechanistic insights into the anti-migratory effects of SDUY429 and SDUY436 on MDA-MB-231 cells. (A) The expression levels of the p GSK3β S9 , p GSK3β Y216 , p AKT S473 and β-catenin proteins were evaluated by Western blotting after treatment with 50 μM SDUY429 or SDUY436 for 24 h; (B) Quantification of relative p AKT S473 protein expression normalized to the control group; (C) Quantification of the relative p GSK3β Y216 protein expression normalized to the control group; (D) Quantification of the relative p GSK3β S9 protein expression normalized to the control group; (E) Quantification of the relative β-catenin nuclear expression normalized to the control group; (F) Quantification of the relative β-catenin membrane expression normalized to the control group; (G) Quantification of the total β-catenin protein expression normalized to the control group; (H) Schematic representation of the anti-migratory signaling pathways involving the hERG channel activation and the AKT/GSK3β/β-catenin axis. All data are presented as mean ± SEM from at least three independent experiments. Statistical significance is denoted as follows: *p < 0.05; **p < 0.01; ***p < 0.001, ****p < 0.0001 versus the control group, ns = not significant.

Article Snippet: The alexa fluor 594 conjugated anti-rabbit secondary antibody (Cat. # bs-0295G-AF594, RRID: AB_2940825) and p GSK3β Y216 (Cat. # bs-4079R, RRID: AB_11070412) antibody were obtained from Bioss (China).

Techniques: Expressing, Western Blot, Control, Membrane, Activation Assay

Journal: Journal of Advanced Research

Article Title: Astragaloside II, a natural saponin, facilitates remyelination in demyelination neurological diseases via p75NTR receptor mediated β-catenin/Id2/MBP signaling axis in oligodendrocyte precursor cells

doi: 10.1016/j.jare.2025.04.028

Figure Lengend Snippet: AS-II inactivates the Wnt/β-catenin signaling pathway in MO3.13 cells and the corpus callosum of CPZ-induced mice . (A) Western blot analysis and quantification of p-GSK3β Ser9 , p-GSK3β Tyr216 , p-β-catenin, TCF4 and Id2 in MO3.13 cell lysates (n = 4). (B) qPCR analysis of mRNA expression levels of TCF4 , Id2 and Olig2 in MO3.13 cells after 72 h AS-II treatment (n = 5). (C) Protein expression levels of p-GSK3β Ser9 , p-GSK3β Tyr216 , p-β-catenin, TCF4, and Id2, and quantification after AS-II administration in mice induced by CPZ (n = 4). (D) Wnt/β-catenin activator SKL2001 counteracted the promoting effect of AS-II on the Wnt/β-catenin signaling pathway in MO3.13 cells (n = 4). Data are presented as mean ± SD; * p < 0.05, ** p < 0.01, *** p < 0.001, vs CPZ group; # p < 0.05, ## p < 0.01, ### p < 0.001, vs Ctrl group; & p < 0.05, &&& p < 0.001, vs AS-II-treated group. Statistical test: One-way ANOVA with Dunnett's test.

Article Snippet: Primary antibodies against glycogen synthase kinase 3β (GSK3β, Cat. No. A11731), p-GSK3β Ser9 (Cat. No. AP1088), p-GSK3β Tyr216 (Cat. No. AP0261), p-β-catenin (Cat. No. A1076), β-catenin (Cat. No. A19657), T-cell factor 4 (TCF4, Cat. No. A19548), Id2 (Cat. No. A0996), and FLAG-Tag (Cat. No. AE063) were provided by ABclonal Technology Co., Ltd. (Wuhan, China).

Techniques: Western Blot, Expressing

Journal: Journal of Advanced Research

Article Title: Astragaloside II, a natural saponin, facilitates remyelination in demyelination neurological diseases via p75NTR receptor mediated β-catenin/Id2/MBP signaling axis in oligodendrocyte precursor cells

doi: 10.1016/j.jare.2025.04.028

Figure Lengend Snippet: Affinity studies between AS-II and its target p75NTR . (A, B) Coomassie Blue staining and MS-based DARTs sequencing were conducted to identify the target proteins of AS-II. (C) Molecular docking visualization of AS-II binding to p75NTR complex structure. (D) Molecular docking results for p75NTR (PDB: 5ZGG) with AS-II, NSC49652, and K252a. (E) Antiproteolytic effect of p75NTR protein in combination with AS-II (n = 4). (F) Effects of varying concentrations of AS-II on its binding and protease resistance to p75NTR (n = 4). (G) Thermal stability of p75NTR with or without AS-II (n = 4). (H) Binding affinities of AS-II and K252a (at concentrations from 1.5625-100 μM), PD90780 (at concentrations from 1.5625-200 μM) to p75NTR were determined using SPR assay. (I) Predicted binding sites of AS-II to p75NTR. (J) Effects of AS-II (100 μM) on the expression of GSK3β Tyr216 , β-catenin, and MBP in MO3.13 cells transfected with WT or mutant p75NTR (n = 4). Data are presented as mean ± SD; & p < 0.05, && p < 0.01, vs GAPDH band; # p < 0.05, ### p < 0.001, vs Ctrl group; * p < 0.05, ** p < 0.01, vs DMSO group. ns, not significant. Statistical test: Two-group comparisons used Student's t -test (E, G, J), multi-group comparisons used one-way ANOVA with Dunnett's test (F). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Article Snippet: Primary antibodies against glycogen synthase kinase 3β (GSK3β, Cat. No. A11731), p-GSK3β Ser9 (Cat. No. AP1088), p-GSK3β Tyr216 (Cat. No. AP0261), p-β-catenin (Cat. No. A1076), β-catenin (Cat. No. A19657), T-cell factor 4 (TCF4, Cat. No. A19548), Id2 (Cat. No. A0996), and FLAG-Tag (Cat. No. AE063) were provided by ABclonal Technology Co., Ltd. (Wuhan, China).

Techniques: Staining, Sequencing, Binding Assay, SPR Assay, Expressing, Transfection, Mutagenesis

Journal: Journal of Advanced Research

Article Title: Astragaloside II, a natural saponin, facilitates remyelination in demyelination neurological diseases via p75NTR receptor mediated β-catenin/Id2/MBP signaling axis in oligodendrocyte precursor cells

doi: 10.1016/j.jare.2025.04.028

Figure Lengend Snippet: Effect of p75NTR knockdown on the promotion of OPC differentiation and maturation by AS-II. MO3 . 13 cells were infected with lentivirus sh-NC or sh-p75NTR to establish a stable transfected cell line and treated with or without AS-II (100 μM) for 72 h. (A) Impact of p75NTR knockdown on AS-II-regulated MBP expression (n = 4). Scale bar: 20 μm. (B) mRNA expression levels of p75NTR , TCF4 , Id2 , and MBP by qPCR assay (n = 5). (C) Western blot analysis and quantification of p75NTR, p-GSK3β Tyr216 , p-β-catenin, TCF4, Id2, and MBP in sh-NC or sh-p75NTR group upon AS-II treatment (n = 4). Data are presented as mean ± SD; * p < 0.05, ** p < 0.01, *** p < 0.001, vs sh-NC DMSO-treated group； ### p < 0.001, vs sh-NC AS-II-treated group; ns, not significant. Statistical test: Student's t -test.

Article Snippet: Primary antibodies against glycogen synthase kinase 3β (GSK3β, Cat. No. A11731), p-GSK3β Ser9 (Cat. No. AP1088), p-GSK3β Tyr216 (Cat. No. AP0261), p-β-catenin (Cat. No. A1076), β-catenin (Cat. No. A19657), T-cell factor 4 (TCF4, Cat. No. A19548), Id2 (Cat. No. A0996), and FLAG-Tag (Cat. No. AE063) were provided by ABclonal Technology Co., Ltd. (Wuhan, China).

Techniques: Knockdown, Infection, Transfection, Expressing, Western Blot

Journal: Neurochemical Research

Article Title: Age- and Sex-Associated Wnt Signaling Dysregulation is Exacerbated from the Early Stages of Neuropathology in an Alzheimer’s Disease Model

doi: 10.1007/s11064-024-04224-7

Figure Lengend Snippet: GSK3β is activated during aging and in the AD model. Representative Western blots and densitometry analysis results showing the protein levels of p-S9GSK3β (inhibitory residue) ( A ), p-Tyr216 (activation residue) ( B ) and total GSK3β ( C ). An age-dependentdownregulation of p-S9GSK3β was observed along with an upregulation of p-Tyr216 in WT animals. These changes occurred earlier in the 3xTg-AD female mice than in the control mice, but there were no significant changes in total GSK3β. The bar graphs show the mean ± SEM from at least 3–6 independent experiments. (* p < 0.05, ** p < 0.01, two-way ANOVA)

Article Snippet: The membranes were blocked with 5% nonfat milk/PBS and incubated with one of the following primary antibodies at 4 °C overnight in 5% nonfat milk/PBS: Dkk-1 (1:500; Abcam Cat #ab61275), Wnt7a/b (1:250; Abcam Cat #ab100792), tau p-S202/Thr205 (1:1000; Thermo Scientific Cat # MN1020), BDNF (1:250; Abcam Cat #ab203573), LRP6 (1:1000; Santa Cruz Biotechnology Cat #sc-25317), p-S9 GSK3β (1:1000 Cell Signaling Technology Cat #9336), p-Tyr216 GSK3β (1:1000; Bio-Rad Cat #AHP2627), GSK3β total (1:1000 Cell Signaling Technology Cat #9315).

Techniques: Western Blot, Residue, Activation Assay, Control

Journal: Life Science Alliance

Article Title: Mitochondrial translation failure represses cholesterol gene expression via Pyk2-Gsk3β-Srebp2 axis

doi: 10.26508/lsa.202302423

Figure Lengend Snippet: (A) Expression of p-Gsk3β (Tyr216) and Gsk3β in the brains of 6-wk-old p32cKO mice and wild-type mice was analyzed by the WB method. The expression level of α-tubulin was used as a control. In addition, the rate of phosphorylation of Tyr216 in Gsk3β was quantified and visualized graphically. In both cases, values were corrected using the results of α-tubulin quantification. Error bars represent mean ± SD. t test was performed to compare WT (n = 7) versus p32cKO (n = 8), **** P < 0.0001. (B) CAP (100 μg/ml) was added to undifferentiated SH-SY5Y cells, and the phosphorylation of Gsk3β at Tyr216 over time and changes in Gsk3β were analyzed by the Western blotting. nSrebp2 and β-actin expression level was used as a control. The right panel shows quantification the phosphorylation rate of Tyr216 in GSK3β. Triplicated experience was performed. t test was performed to compare WT cells versus WT cells treated with CAP, * P < 0.05, ** P < 0.01, *** P < 0.001. (C) CAP (100 μg/ml) was added to differentiated SH-SY5Y cells, and the phosphorylation of Gsk3β at Tyr216 over time and changes in Gsk3β were analyzed by the Western blotting. nSrebp2 and β-actin expression level was used as a control. The right panel shows quantification the phosphorylation rate of Tyr216 in Gsk3β. Triplicated experience was performed. (D) Gsk3β activity levels were measured in the brains of 6-wk-old p32cKO mice and wild-type mice. Error bars represent mean ± SD. t test was performed to compare WT (n = 3) versus p32cKO (n = 3), * P < 0.05. (E) Expression of cholesterol synthase genes in SH-SY5Y cells treated with CAP (100 μg/ml) or GSK3β inhibitor (tideglusib) (20 μM), or left untreated was analyzed using real-time PCR. The treatment with GSK3β inhibitor and CAP was performed for 48 h. The expression level of 18S rRNA was measured as a control. Error bars represent mean ± SD of three independent experiments. (F) Proteins were extracted from SH-SY5Y cells treated with CAP (100 μg/ml) or GSK3β inhibitor (CHIR-99021) (20 μM), or left untreated and analyzed for nSrebp2 expression using the Western blotting. β-Actin expression level was used as a control. A triple experience was carried out. The right panel shows quantification the phosphorylation rate of Tyr216 in Gsk3β. Source data are available for this figure.

Article Snippet: We used the following primary antibodies for Western blotting: Srebp2 (10007663; Cayman Chemical), Gsk3β (9315P; Cell Signaling), Gsk3β [pTyr216] (NB100-81946; Novus Biologicals), Gsk3β [pSer9] (9323; Cell Signaling), Pyk2 (3292S; Cell Signaling), Pyk2 [pTyr402] (3291S; Cell Signaling), COX1 (459600; Novex by life technologies), SDHA (459200; Invitrogen), β-actin (#A5441; Sigma-Aldrich), α-Tubulin (ab7291; Abcam), Scap (13102; Cell Signaling), Srebp1 (ab28481; Abcam).

Techniques: Expressing, Control, Phospho-proteomics, Western Blot, Activity Assay, Real-time Polymerase Chain Reaction

Journal: Life Science Alliance

Article Title: Mitochondrial translation failure represses cholesterol gene expression via Pyk2-Gsk3β-Srebp2 axis

doi: 10.26508/lsa.202302423

Figure Lengend Snippet: (A) CAP (100 μg/ml) was added to undifferentiated MEF cells, and the phosphorylation of Gsk3β at Tyr216 over time and changes in Gsk3β, nSrebp2 were analyzed by the Western blotting. β-Actin expression level was used as a control. The right panel shows quantification the phosphorylation rate of Tyr216 in Gsk3β. Triplicated experience was performed. t test was performed to compare WT cells versus WT cells treated with CAP, * P < 0.05, ** P < 0.01. (B) Proteins were extracted from SH-SY5Y cells treated with CAP (100 μg/ml) or GSK3β inhibitor (tideglusib) (100 μM), or left untreated and analyzed for Gsk3β phosphorylation (Ser9) (n = 1). β-Actin expression level was used as a control. (C) Proteins were extracted from SH-SY5Y cells treated with CAP (100 μg/ml) or GSK3β inhibitor (tideglusib) (100 μM), or left untreated and analyzed for nSREBP1 expression using the Western blotting. β-Actin expression level was used as a control. The right panel shows quantification of nSrebp1. Triplicated experience was performed. Source data are available for this figure.

Article Snippet: We used the following primary antibodies for Western blotting: Srebp2 (10007663; Cayman Chemical), Gsk3β (9315P; Cell Signaling), Gsk3β [pTyr216] (NB100-81946; Novus Biologicals), Gsk3β [pSer9] (9323; Cell Signaling), Pyk2 (3292S; Cell Signaling), Pyk2 [pTyr402] (3291S; Cell Signaling), COX1 (459600; Novex by life technologies), SDHA (459200; Invitrogen), β-actin (#A5441; Sigma-Aldrich), α-Tubulin (ab7291; Abcam), Scap (13102; Cell Signaling), Srebp1 (ab28481; Abcam).

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

Journal: Life Science Alliance

Article Title: Mitochondrial translation failure represses cholesterol gene expression via Pyk2-Gsk3β-Srebp2 axis

doi: 10.26508/lsa.202302423

Figure Lengend Snippet: (A) The expression of p-Pyk2 (Tyr402) and Pyk2 in the brains of 6-wk-old p32cKO mice (n = 8) and wild-type mice (n = 7) was analyzed by the Western blotting. The expression level of α-tubulin was used as a control. The rate of phosphorylation of Tyr402 in Pyk2 was quantified and visualized graphically. Both values were corrected using the results of α-tubulin quantification. Error bars represent mean ± SD. t test was performed to compare WT (n = 7) versus p32cKO (n = 8), **** P < 0.0001. (B) CAP (100 μg/ml) was added to undifferentiated SH-SY5Y cells, and changes in Pyk2 phosphorylation of Tyr402, Pyk2, Gsk3β phosphorylation of Tyr216, and Gsk3β protein expression over time were analyzed by the Western blotting. The expression level of β-actin was used as a control. A triple experience was carried out. The right panel shows quantification the phosphorylation rate of Tyr402 in Pyk2 and Tyr216 in Gsk3β. (C) CAP (100 μg/ml) was added to differentiated SH-SY5Y cells, and changes in Pyk2 phosphorylation of Tyr402 and Pyk2 protein expression over time were analyzed by the Western blotting. The expression level of β-actin was used as a control. A triple experience was carried out. The right panel shows quantification the phosphorylation rate of Tyr402 in Pyk2. (D) Proteins were extracted from SH-SY5Y cells treated with CAP (100 μg/ml) for 3 h or PYK2 inhibitor (PF-562271) (100 μM) for 3 h, or left untreated and analyzed for p-Gsk3β, Gsk3β, p-Pyk2, and Pyk2 expression using the WB method. β-Actin expression level was used as a control. A triple experience was carried out. The right panel shows quantification the phosphorylation rate of Tyr402 in Pyk2 and Tyr216 in Gsk3β. * showed non-specific band. (E) Proteins were extracted from SH-SY5Y cells treated with CAP (100 μg/ml) for 72 h or PYK2 inhibitor (PF-562271) (1 μM) for 72 h, or left untreated and analyzed for nSrebp2 expression using the Western blotting. β-Actin expression level was used as a control. A triple experience was carried out. The right panel shows the quantification of nSrebp2. Source data are available for this figure.

Article Snippet: We used the following primary antibodies for Western blotting: Srebp2 (10007663; Cayman Chemical), Gsk3β (9315P; Cell Signaling), Gsk3β [pTyr216] (NB100-81946; Novus Biologicals), Gsk3β [pSer9] (9323; Cell Signaling), Pyk2 (3292S; Cell Signaling), Pyk2 [pTyr402] (3291S; Cell Signaling), COX1 (459600; Novex by life technologies), SDHA (459200; Invitrogen), β-actin (#A5441; Sigma-Aldrich), α-Tubulin (ab7291; Abcam), Scap (13102; Cell Signaling), Srebp1 (ab28481; Abcam).

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

Journal: Life Science Alliance

Article Title: Mitochondrial translation failure represses cholesterol gene expression via Pyk2-Gsk3β-Srebp2 axis

doi: 10.26508/lsa.202302423

Figure Lengend Snippet: (A) CAP (100 μg/ml) was added to differentiated Oligodendrocyte cells, and changes in Pyk2 phosphorylation of Tyr402 and Pyk2 protein expression over time were analyzed by the Western blotting. The expression level of β-actin was used as a control. A triple experience was carried out. The right panel shows quantification the phosphorylation rate of Tyr402 in Pyk2. t test was performed to compare WT cells versus WT cells treated with CAP, * P < 0.05, ** P < 0.01. (B) Proteins were extracted from SH-SY5Y cells treated with CAP (100 μg/ml) or GSK3β inhibitor (CHIR-99021) (20 μM), or left untreated and analyzed for Pyk2 phosphorylation level [p-Pyk2 (Thy402)/Pyk2] and Gsk3β phosphorylation level [p-Gsk3β(Thy216)/Gsk3β]. β-Actin expression level was used as a control. A triple experience was carried out. Source data are available for this figure.

Article Snippet: We used the following primary antibodies for Western blotting: Srebp2 (10007663; Cayman Chemical), Gsk3β (9315P; Cell Signaling), Gsk3β [pTyr216] (NB100-81946; Novus Biologicals), Gsk3β [pSer9] (9323; Cell Signaling), Pyk2 (3292S; Cell Signaling), Pyk2 [pTyr402] (3291S; Cell Signaling), COX1 (459600; Novex by life technologies), SDHA (459200; Invitrogen), β-actin (#A5441; Sigma-Aldrich), α-Tubulin (ab7291; Abcam), Scap (13102; Cell Signaling), Srebp1 (ab28481; Abcam).

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