Journal: Journal of Biochemical and Molecular Toxicology

Article Title: Eupatilin Alleviates Acute Myocardial Ischemia‐Reperfusion Injury in Rats via the Akt/GSK‐3β Pathway by Targeting COXs

doi: 10.1002/jbt.71111

Figure Lengend Snippet: Antibodies in this study.

Article Snippet: Phospho‐GSK‐3β‐S9 , AP0039 , WB: 1:300 , Abclonal.

Techniques:

Journal: Journal of Biochemical and Molecular Toxicology

Article Title: Eupatilin Alleviates Acute Myocardial Ischemia‐Reperfusion Injury in Rats via the Akt/GSK‐3β Pathway by Targeting COXs

doi: 10.1002/jbt.71111

Figure Lengend Snippet: Antibodies in this study.

Article Snippet: GSK‐3β , A2081 , WB: 1:1000 , Abclonal.

Techniques:

Journal: Journal of Biochemical and Molecular Toxicology

Article Title: Eupatilin Alleviates Acute Myocardial Ischemia‐Reperfusion Injury in Rats via the Akt/GSK‐3β Pathway by Targeting COXs

doi: 10.1002/jbt.71111

Figure Lengend Snippet: Effects of eupatilin on the COX‐mediated Akt/GSK pathway in OGD/R‐induced H9c2 cells. The levels of COX1 (A), COX2 (B), phosphorylated Akt1 (S473), and phosphorylated GSK‐3β (S9) proteins (C) were measured by Western blotting. (D) The interaction between eupatilin and COXs was predicted using molecular docking. n = 6, * p < 0.05, ** p < 0.01.

Article Snippet: GSK‐3β , A2081 , WB: 1:1000 , Abclonal.

Techniques: Western Blot, Docking Assay

Journal: Journal of Biochemical and Molecular Toxicology

Article Title: Eupatilin Alleviates Acute Myocardial Ischemia‐Reperfusion Injury in Rats via the Akt/GSK‐3β Pathway by Targeting COXs

doi: 10.1002/jbt.71111

Figure Lengend Snippet: Eupatilin treatment upregulated the Akt/GSK pathway via COXs in OGD/R‐induced H9c2 cells. Levels of COX1 (A), COX2 (B), p‐Akt1 (S473)/Akt1, and p‐GSK‐3β (S9)/GSK‐3β (C) proteins were measured by western blotting. n = 6, * p < 0.05, ** p < 0.01.

Article Snippet: GSK‐3β , A2081 , WB: 1:1000 , Abclonal.

Techniques: Western Blot

Journal: Journal of Biochemical and Molecular Toxicology

Article Title: Eupatilin Alleviates Acute Myocardial Ischemia‐Reperfusion Injury in Rats via the Akt/GSK‐3β Pathway by Targeting COXs

doi: 10.1002/jbt.71111

Figure Lengend Snippet: Eupatilin inhibited the COXs and upregulated the Akt/GSK pathway in MIRI‐induced rats. The levels of COX1 (A), COX2 (B), p‐Akt1 (S473)/Akt1, and p‐GSK‐3β (S9)/GSK‐3β (C) proteins were measured by Western blotting. n = 6, * p < 0.05, ** p < 0.01.

Article Snippet: GSK‐3β , A2081 , WB: 1:1000 , Abclonal.

Techniques: Western Blot

Journal: Science Advances

Article Title: GSK3β and Plk1 sequentially phosphorylate ATP–citrate lyase to promote homologous recombination

doi: 10.1126/sciadv.aeg1097

Figure Lengend Snippet: ( A and B ) HeLa cells were synchronized in G 2 phase using a double-thymidine block. Seven hours after release, cells were treated with roscovitine or DMSO (control) for 2 hours and then either immunoblotted (A) or subjected to pull-down with the indicated GST fusion proteins, followed by immunoblotting and CBB staining (B). Irrelevant lanes were excised (indicated by a vertical line). ( C ) Asynchronous or nocodazole-arrested mitotic HeLa cells were subjected to pull-down with the indicated proteins, followed by immunoblotting and CBB staining. ( D ) Asynchronous or nocodazole-arrested mitotic HeLa cells expressing HA-Plk1 and/or SFB-ACLY were subjected to IP with anti-Flag beads, followed by immunoblotting. ( E and F ) HeLa cells were treated with BI2536, GSK461364, or DMSO (control) for 3 hours. Cells were then either immunoblotted (E) or subjected to pull-down with the indicated proteins, followed by immunoblotting and CBB staining (F). ( G and H ) HeLa cells were transfected with control or GSK3β siRNA. Forty-eight hours later, cells were either immunoblotted (G) or subjected to pull-down with the indicated proteins, followed by immunoblotting and CBB staining (H). ( I and J ) HeLa cells were transfected with control or GSK3α siRNA. Forty-eight hours later, cells were either immunoblotted (I) or subjected to pull-down with the indicated proteins, followed by immunoblotting and CBB staining (J). ( K ) SFB-ACLY immunoprecipitated from HEK293T cells pretreated with LY2090314 for 3 hours was subjected to in vitro phosphorylation with recombinant 6×His-GSK3β in the presence or absence of ATP, followed by immunoblotting with the antibodies against GSK3β, Flag tag, and ACLY-pT447. ( L ) SFB-ACLY was immunoprecipitated and phosphorylated as in (K) and then analyzed by immunoblotting and Far-Western blotting. See also fig. S3.

Article Snippet: Primary antibodies used were as follows: ACLY (Abcam, ab40793), Flag tag (GenScript, A01868; for immunoblotting), GST (Sigma-Aldrich, G7781), HA tag (BioLegend, 16B12), Myc-tag (05-724, Millipore), MBP (New England BioLabs, E8032), pan-pSer/Thr (ABclonal, AP0893), pan-Ser-pThr-Pro (Cell Signaling Technology, D73F6; 5243), GSK3α (ABclonal, A19060), GSK3β (ABclonal, A6164), ACLY-pS455 (Cell Signaling Technology, 4331), BRCA1 (Santa Cruz Biotechnology, sc-6954), γH2AX (Abcam, ab81299), cyclin A2 (ABclonal, A19036), cyclin A2 (Proteintech, 66391-1-Ig), 53BP1 (made in-house), pan-AKT (ABclonal, A18120), RAD51 (made in-house), histone H4 (Sigma-Aldrich, 07-108), H4K16ac (Abcam, ab109463), α-tubulin (ABclonal, AC012), GFP (Abmart, M20004), Plk1 (Abcam, ab17057), and Plk1 phospho-T210 (BD Transduction, 558400).

Techniques: Blocking Assay, Control, Western Blot, Staining, Expressing, Transfection, Immunoprecipitation, In Vitro, Phospho-proteomics, Recombinant, FLAG-tag, Far Western Blot

Journal: Science Advances

Article Title: GSK3β and Plk1 sequentially phosphorylate ATP–citrate lyase to promote homologous recombination

doi: 10.1126/sciadv.aeg1097

Figure Lengend Snippet: ( A ) Schematic of ACLY residues Ser 451 , Ser 455 , and Ser 459 as potential priming sites for GSK3β-mediated phosphorylation of Thr 447 . Dashed arrows denote possible priming effect. ( B ) HEK293T cells expressing SFB-ACLY (WT, T447A, S451A, or S455A), or an empty vector control, were analyzed by immunoblotting. ( C and D ) HEK293T cells expressing the indicated ACLY proteins were subjected to pull-down with the indicated GST fusion proteins, followed by immunoblotting for SFB-ACLY (WT or S451A) (C) and SFB-ACLY (WT or S455A) (D) and CBB staining. ( E ) SFB-ACLY (WT, S455A, or S451A) immunoprecipitated from HEK293T cells pretreated with LY2090314 for 3 hours was subjected to in vitro phosphorylation with recombinant 6×His-GSK3β, followed by immunoblotting. ( F ) HeLa cells were treated with DMSO (control) or MK-2206 for 3 hours and analyzed by immunoblotting. ( G ) HeLa cells were transfected with control siRNA or two distinct siRNAs targeting AKT1. Cells were harvested 48 hours later and analyzed by immunoblotting. ( H ) HEK293T cells expressing ACLY-GFP (WT, S442A, or S455A) were subjected to IP with anti-GFP beads, followed by immunoblotting. ( I ) Immunoblot analysis of whole-cell lysates from parental HeLa cells and HeLa cells stably expressing ACLY-GFP (WT or S442A). See also fig. S10.

Article Snippet: Primary antibodies used were as follows: ACLY (Abcam, ab40793), Flag tag (GenScript, A01868; for immunoblotting), GST (Sigma-Aldrich, G7781), HA tag (BioLegend, 16B12), Myc-tag (05-724, Millipore), MBP (New England BioLabs, E8032), pan-pSer/Thr (ABclonal, AP0893), pan-Ser-pThr-Pro (Cell Signaling Technology, D73F6; 5243), GSK3α (ABclonal, A19060), GSK3β (ABclonal, A6164), ACLY-pS455 (Cell Signaling Technology, 4331), BRCA1 (Santa Cruz Biotechnology, sc-6954), γH2AX (Abcam, ab81299), cyclin A2 (ABclonal, A19036), cyclin A2 (Proteintech, 66391-1-Ig), 53BP1 (made in-house), pan-AKT (ABclonal, A18120), RAD51 (made in-house), histone H4 (Sigma-Aldrich, 07-108), H4K16ac (Abcam, ab109463), α-tubulin (ABclonal, AC012), GFP (Abmart, M20004), Plk1 (Abcam, ab17057), and Plk1 phospho-T210 (BD Transduction, 558400).

Techniques: Phospho-proteomics, Expressing, Plasmid Preparation, Control, Western Blot, Staining, Immunoprecipitation, In Vitro, Recombinant, Transfection, Stable Transfection