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: 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