Journal: bioRxiv

Article Title: Mechanistic insight into the phosphorylation of ERK by MEK

doi: 10.64898/2026.03.13.710243

Figure Lengend Snippet: a , b , pMEK1-catalyzed dephosphorylation of pY-ERK1. In an ATP-, ADP-, or AMP-PNP-containing buffer, or a nucleotide-free buffer, pY-ERK1 was incubated with various concentrations of pMEK1 at 30, after 3 minutes, the phosphorylation of ERK1 T202 and Y204 was evaluated using Western blotting ( a ) and quantified ( b ). c , d , pMEK1-catalyzed dephosphorylation of pTY-ERK1. In an ADP- or AMP-PNP-containing buffer, or a nucleotide-free buffer, pTY-ERK1 was incubated with pMEK1 at 30. At each time point, the phosphorylation of ERK1 T202 and Y204 was evaluated using Western blotting ( c ) and quantified ( d ). e , f , Effect of mutations of the nucleotide-binding pocket of MEK1 on the activity of pMEK1 to catalyze phosphate group transfer from ERK1 Y204 to T202. In a nucleotide-free buffer, pY-ERK1 was incubated with wild-type (WT) or mutant pMEK1 at 30, and then the phosphorylation of ERK1 T202 and Y204 was evaluated using Western blotting ( e ) and quantified ( f ). The gels in ( a ), ( c ) and ( e ) are the results of a representative experiment out of three independent experiments. The data in ( b ), ( d ) and ( f ) represent the mean ± SD of three independent measurements.

Article Snippet: ATP, ADP (Selleck, Cat# S6325, 100 mM stock in H 2 O) and AMP-PNP (Sigma, Cat# A2647, 100 mM stock in H 2 O) were each prepared as a 4× stock in the reaction buffer.

Techniques: De-Phosphorylation Assay, Incubation, Phospho-proteomics, Western Blot, Binding Assay, Activity Assay, Mutagenesis

Journal: bioRxiv

Article Title: Mechanistic insight into the phosphorylation of ERK by MEK

doi: 10.64898/2026.03.13.710243

Figure Lengend Snippet: a , Summary of the dissociation constants ( K d ) for the binding between MEK1 (uMEK1 or pMEK1) and ERK1 (uERK1 or thio-pTY-ERK1) in the absence or presence of ADP or AMP-PNP. The K d values were measured using Isothermal Titration Calorimetry (ITC). b , Cycle of the pMEK1-catalyzed ERK1 phosphorylation reaction. In addition to the canonical sequential mechanism for pMEK1 (where Y204 and T202 are phosphorylated sequentially by ATP), we proposed a relay mechanism to explain the phosphorylation process of ERK1 T202: Y204 is phosphorylated first, its phosphate group is then transferred to T202, and Y204 is phosphorylated again by ATP to generate dual-phosphorylated ERK1.

Article Snippet: ATP, ADP (Selleck, Cat# S6325, 100 mM stock in H 2 O) and AMP-PNP (Sigma, Cat# A2647, 100 mM stock in H 2 O) were each prepared as a 4× stock in the reaction buffer.

Techniques: Binding Assay, Isothermal Titration Calorimetry, Phospho-proteomics

Journal: bioRxiv

Article Title: Mechanistic insight into the phosphorylation of ERK by MEK

doi: 10.64898/2026.03.13.710243

Figure Lengend Snippet: a , Purified unphosphorylated (uERK1), phosphorylated (pY-ERK1 and pTY-ERK1), and thio-phosphorylated ERK1 (thio-pTY-ERK1) were examined by SDS–PAGE and visualized by Coomassie blue staining. b , Comparison of the ATP consumption rate of thio-pTY-ERK1 with those of uERK1, pY-ERK1, and pTY-ERK1. The ATP consumption was measured using an ADP-Glo Kinase Assay. The MBP peptide FFKNIVTPRTPPPSQGK was used as the substrate of ERK1. The data represent the mean ± SD of three independent measurements. c , d , pMEK1-catalyzed dephosphorylation of pTY-ERK1 ( c ) and thio-pTY-ERK1 ( d ) in an ADP-containing buffer. The percentage of ERK1 in different phosphorylation states was determined by LC-MS.

Article Snippet: ATP, ADP (Selleck, Cat# S6325, 100 mM stock in H 2 O) and AMP-PNP (Sigma, Cat# A2647, 100 mM stock in H 2 O) were each prepared as a 4× stock in the reaction buffer.

Techniques: Purification, SDS Page, Staining, Comparison, Kinase Assay, De-Phosphorylation Assay, Phospho-proteomics, Liquid Chromatography with Mass Spectroscopy

Journal: The EMBO Journal

Article Title: CEP192 localises mitotic Aurora-A activity by priming its interaction with TPX2

doi: 10.1038/s44318-024-00240-z

Figure Lengend Snippet: Reagents and tools table

Article Snippet: ADP , Thermo Scientific Acros , Cat #164670050.

Techniques: Recombinant, Plasmid Preparation, Sequencing, CRISPR, Control, Protease Inhibitor, Transfection, Software, Protein Purification, Mutagenesis, Mass Spectrometry, Western Blot

Journal: eLife

Article Title: Distinct mitochondrial defects trigger the integrated stress response depending on the metabolic state of the cell

doi: 10.7554/eLife.49178

Figure Lengend Snippet:

Article Snippet: The extraction buffer included 0.5 μM Adenosine- 15 N 5 5′-Monophosphate (Sigma; 900382), 1 μM Adenosine- 13 C 5 5’-Diphosphate (Toronto Research Chemicals, North York, Ontario, Canada; A281697), 5 μM Adenosine- 13 C 10 5′-Triphosphate (Sigma; 710695) and 2.5 μM L -Aspartic acid-1,4- 13 C 2 (Cambridge Isotope Laboratories, Tewksbury, MA; CLM-4455).

Techniques: Knock-Out, Recombinant, Luciferase, Expressing, Plasmid Preparation, Modification, Concentration Assay, Gene Expression

Journal: Cell Communication and Signaling : CCS

Article Title: CD73/adenosine axis exerts cardioprotection against hypobaric hypoxia-induced metabolic shift and myocarditis in a sex-dependent manner

doi: 10.1186/s12964-024-01535-8

Figure Lengend Snippet: CD73/adenosine axis mediates immunomodulation during HH in a sex-dependent manner. A - B ELISA analysis of Extracellular Adenosine level and CD73 activity ( n = 4 mice/group). C Relative mRNA levels of adenosine receptors (A1, A2a, A2b, A3 and n = 4mice/group). D-F Representative immunohistochemical staining and graphical presentation of macrophage CD86 and CD206-positive cells assessed from the myocardial sections ( n = 4–6 hearts per group) Scale bar, 50 μm. G-K Graphical presentation of sera cardiac troponin I (cTnI) concentrations and inflammatory cytokines; Interleukin (IL)-1β, IL-18, IL-10, and transforming growth factor (TGF)-β concentrations assessed by ELISA and RT-qPCR using myocardia lysates. All ELISA were performed in triplicates ( n = 5–6 mice per group). M = male; MH = male + HH; F = female; FH = female + HH; MI = male + APCP; FI = female + APCP; MHI = male + HH + APCP; FHI = female + HH + APCP. Data are presented as mean ± SEM; *** p < 0.001; ** p < 0.01; * p < 0.05

Article Snippet: CD73 inhibitor Adenosine 5′-(α, β-methylene) diphosphate sodium salt (APCP) was purchased from Tocris Bio-Techne, and the prostatic acid phosphatase (PAP) inhibitor – Benzylphosphonic acid (GB57232) (BPA) was purchased from GlpBio Technology Inc. All the drugs were prepared according to the manufacturer's instructions.

Techniques: Enzyme-linked Immunosorbent Assay, Activity Assay, Immunohistochemical staining, Staining, Quantitative RT-PCR

Journal: Cell Communication and Signaling : CCS

Article Title: CD73/adenosine axis exerts cardioprotection against hypobaric hypoxia-induced metabolic shift and myocarditis in a sex-dependent manner

doi: 10.1186/s12964-024-01535-8

Figure Lengend Snippet: Hyperactivity of CD73/adenosine axis promotes myocardial metabolic shift in a sex-dependent manner during HH and protein expression of the other ectonucleotidases. A - B Representative Immunofluorescence and graphical presentation of the co-staining of F4/80 and Glut1 with nuclei (DAPI). Color channels were adjusted in the merged images to enhance the visualization of all the respective fluorescence dyes. Scale bar, 50 μm. C - E Representative Oil Red O (ORO) and Periodic Acid Schiff (PAS) staining of myocardial sections and their respective graphical presentations showing lipid and glycogen deposition percentages ( n = 4–6 sections per 4–6 mice per group) Scale bar, 50 μm. F - H Representative Immunoblotting of CD36 and Glut1 and their respective Graphical plots; each blot band in the representative blot is an independent biological sample ( n = 3 hearts per group). I - L Representative Immunoblotting of CD73, TNAP, and PAP and their respective Graphical plots; each blot band in the representative blot is an independent biological sample ( n = 3 hearts per group). M = male; MH = male + HH; F = female; FH = female + HH; MI = male + APCP; FI = female + APCP; MHI = male + HH + APCP; FHI = female + HH + APCP. Data are expressed as mean ± SEM; * p < 0.05, ** p < 0.01, *** p < 0.001

Article Snippet: CD73 inhibitor Adenosine 5′-(α, β-methylene) diphosphate sodium salt (APCP) was purchased from Tocris Bio-Techne, and the prostatic acid phosphatase (PAP) inhibitor – Benzylphosphonic acid (GB57232) (BPA) was purchased from GlpBio Technology Inc. All the drugs were prepared according to the manufacturer's instructions.

Techniques: Expressing, Immunofluorescence, Staining, Fluorescence, Western Blot

Journal: Cell Communication and Signaling : CCS

Article Title: CD73/adenosine axis exerts cardioprotection against hypobaric hypoxia-induced metabolic shift and myocarditis in a sex-dependent manner

doi: 10.1186/s12964-024-01535-8

Figure Lengend Snippet: Double inhibition of PAP and CD73 in male mice, mitigated glycolytic shift and proinflammatory response. A - C Representative Immunoblotting of CD73, TNAP, and PAP and their respective Graphical plots; each blot band in the representative blot is an independent biological sample ( n = 3 hearts per group). E ELISA analysis of Extracellular Adenosine level (n = 4 mice/group). F - H Representative Oil Red O (ORO) and Periodic Acid Schiff (PAS) staining of myocardial sections and their respective graphical presentations showing lipid and glycogen deposition percentages ( n = 4–6 sections per 4–5 mice per group) Scale bar, 50 μm. I - K Representative Immunoblotting of CD36 and Glut1 and their respective Graphical plots; each blot band in the representative blot is an independent biological sample ( n = 3 hearts per group). L - N Representative immunohistochemical staining and graphical presentation of macrophage CD86 and CD206-positive cells assessed from the myocardial sections ( n = 4–5 hearts per group) Scale bar, 50 μm. O - Q Inflammatory cytokines; Interleukin TNF-α, IL-10, and transforming growth factor (TGF)-β concentrations assessed by ELISA using myocardia lysates. All ELISA were performed in triplicates ( n = 4–5 mice per group). M = male; MH = male + HH; F = female; FH = female + HH; MI = male + APCP; FI = female + APCP; MHI = male + HH + APCP; FHI = female + HH + APCP. Data are expressed as mean ± SEM. * p < 0.05, ** p < 0.01, *** p < 0.001

Article Snippet: CD73 inhibitor Adenosine 5′-(α, β-methylene) diphosphate sodium salt (APCP) was purchased from Tocris Bio-Techne, and the prostatic acid phosphatase (PAP) inhibitor – Benzylphosphonic acid (GB57232) (BPA) was purchased from GlpBio Technology Inc. All the drugs were prepared according to the manufacturer's instructions.

Techniques: Inhibition, Western Blot, Enzyme-linked Immunosorbent Assay, Staining, Immunohistochemical staining

Journal: iScience

Article Title: MAPK/ERK-PK(Ser11) pathway regulates divergent thermal metabolism of two congeneric oyster species

doi: 10.1016/j.isci.2024.110321

Figure Lengend Snippet:

Article Snippet: Adenosine 5'-diphosphate , MCE , Cat#HY-W010918.

Techniques: Virus, Recombinant, Pyruvate Assay, Magnetic Beads, Lysis, Western Blot, Protease Inhibitor, Software