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: Experimental & Molecular Medicine

Article Title: Adenosine transmission from hypothalamic tanycytes to AGRP/NPY neurons regulates energy homeostasis

doi: 10.1038/s12276-025-01449-6

Figure Lengend Snippet: a A schematic diagram of ATP release from tanycytes and conversion of nucleotides by ectonucleotidases. ATP is released through Cx43 from tanycytes and is converted to ADP and AMP by ENTPD. AMP is converted to adenosine by NT5E. Adenosine is transported by ENT1 and is converted to inosine by ADA. b Relative expression levels of ectonucleotidases families and nucleoside transporters in A2/29 cells ( n = 6). c Relative intracellular and extracellular adenosine levels in si-scram and si- Tspo A2/29 cells ( n = 9 per group). d , f Relative intracellular and extracellular ATP levels in si-scram and si- Tspo A2/29 cells in the presence or absence of ENTPD inhibitor ( ARL67156 , 0.1 mM) ( d ) or NT5E inhibitor (APCP, 0.1 mM) ( f ) for 3 h ( n = 9 per group). e, g – i Relative intracellular and extracellular adenosine levels in si-scram and si- Tspo A2/29 cells in the presence or absence of ARL67156 ( e ) APCP ( g ) ADA inhibitor (EHNA, 0.01 mM) ( h ) or ENT1 inhibitor (NBMPR, 0.01 mM) ( i ) for 3 h ( n = 9 per group). Data are mean ± s.e.m. or boxes indicating the interquartile range with whiskers, and dotted lines indicate that significance was calculated separately for each. Significance was determined by one-way ANOVA with Dunnett’s multiple-comparisons test (** P < 0.01, **** P < 0.0001) in b two-tailed unpaired Student’s t -test (*** P < 0.001) in c or otherwise by two-way ANOVA with Šidák’s multiple-comparisons test (** P < 0.01, *** P < 0.001, **** P < 0.0001). n.s., not significant.

Article Snippet: A2/29 cells were treated with Gap26 (0.05 mM), ARL67156 (0.1 mM), APCP (0.1 mM; Tocris, 3633), EHNA (0.01 mM; Tocris, 1261), NBMPR (0.01 mM; Tocris, 0625) or vehicle.

Techniques: Expressing, Two Tailed Test

Journal: Experimental & Molecular Medicine

Article Title: Adenosine transmission from hypothalamic tanycytes to AGRP/NPY neurons regulates energy homeostasis

doi: 10.1038/s12276-025-01449-6

Figure Lengend Snippet: a , d , g , j Schematic illustrations of ARL67156 ( a ) APCP ( d ) EHNA ( g ) and NBMPR ( j ) treatments in the indirect coculture system. b , e , h , k Relative extracellular ATP or adenosine levels in si-scram or si- Tspo A2/29 cells indirectly cocultured with N41 cells in the presence or absence of ARL67156 ( b ) APCP ( e ) EHNA ( h ) or NBMPR ( k ) under 0.06 mM oleic acid (OL) treatment for 3 h ( n = 9 per group). c , f , i , l Relative mRNA levels of Agrp and Npy in N41 cells cocultured with si-scram or si- Tspo A2/29 cells in the presence or absence of ARL67156 ( c ), APCP ( f ) EHNA ( i ) or NBMPR ( l ) under OL treatment for 3 h ( n = 9 per group). Data are mean ± s.e.m. or boxes indicating the interquartile range with whiskers, and dotted linesindicate that significance was calculated separately for each. Significance was determined by two-way ANOVA with Šidák’s multiple-comparisons test (* P < 0.5, ** P < 0.01, *** P < 0.001, **** P < 0.0001; n.s., not significant).

Article Snippet: A2/29 cells were treated with Gap26 (0.05 mM), ARL67156 (0.1 mM), APCP (0.1 mM; Tocris, 3633), EHNA (0.01 mM; Tocris, 1261), NBMPR (0.01 mM; Tocris, 0625) or vehicle.

Techniques:

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