Journal: Ecotoxicology and environmental safety

Article Title: PRKAA1 induces aberrant mitophagy in a PINK1/Parkin-dependent manner, contributing to fluoride-induced developmental neurotoxicity.

doi: 10.1016/j.ecoenv.2023.114772

Figure Lengend Snippet: Fig. 5. NaF exposure induces elevated phos phorylation of PRKAA1 expression in vivo and in vitro. (A) The hierarchical cluster heatmap of phosphorylated proteins in rat hippocampus. (B) Statistics of different phosphorylation sites and proteins in rat hippocampus. (C) Volcano plot of differentially phosphorylated proteins in rat hippocampus. The criteria for significance were set at logarithmic fold change (log2 FC) of > 1.2 or < 0.8 (P < 0.05). (D) Phosphorylation of PRKAA1 protein in rat hippocampus. (E-F) Representative western blot and relative quan tifications of PRKAA1 and p-PRKAA1 in rat hippocampus. (G-H) Representative western blot and relative quantifications of PRKAA1 and p-PRKAA1 in SH-SY5Y cells. All experiments were performed independently and repeated 3 times. The data were presented as the means ± SD. *P < 0.05 compare with the control group.

Article Snippet: PRKAA1, p-PRKAA1 (Thr172), Parkin, and Cyt C antibodies were obtained from Boster Biotech (China).

Techniques: Expressing, In Vivo, In Vitro, Phospho-proteomics, Western Blot, Control

Journal: Ecotoxicology and environmental safety

Article Title: PRKAA1 induces aberrant mitophagy in a PINK1/Parkin-dependent manner, contributing to fluoride-induced developmental neurotoxicity.

doi: 10.1016/j.ecoenv.2023.114772

Figure Lengend Snippet: Fig. 6. DM inhibits aberrant mitophagy by pre venting the phosphorylation of PRKAA1 in vitro. (A-B) Representative western blot and relative quantifications of PRKAA1and p-PRKAA1 in SH- SY5Y cells. (C-D) Representative western blot and relative quantifications of PINK1 and Parkin in SH-SY5Y cells. (E-F) Representative images and relative quantifications of immunofluores cence staining of PINK1 and Mito-tracker, after DM intervention. The scale bar represents 50 µm. (G-H) Representative western blot and relative quantifications of TOMM-20 in SH-SY5Y cells. All experiments were performed independently and repeated 3 times. The data were presented as the means ± SD. *P < 0.05 compare with the control group, @P < 0.05 compare with the NaF group.

Article Snippet: PRKAA1, p-PRKAA1 (Thr172), Parkin, and Cyt C antibodies were obtained from Boster Biotech (China).

Techniques: Phospho-proteomics, In Vitro, Western Blot, Staining, Control

Journal: Bioactive materials

Article Title: Calcium phosphate-based materials regulate osteoclast-mediated osseointegration.

doi: 10.1016/j.bioactmat.2021.05.003

Figure Lengend Snippet: Fig. 5. Effect of Ca/P ratio in CPC on RANKL-RANK signaling. (a) Immunofluorescence analysis of RANK expression (red) in osteoclasts on surface of CPC scaffold. (b) Concentration of RANK was analysis by ELISA. The concentration of RANK was measured after inducing BMMs for 1 days. (c) Affinity between RANKL and RANK was measured in BIAcore T200 system. Human-RANK anchored on the chip was applied to interact with the human-RANKL dissolved into the CPC extract. The vehicle means MEM-α medium. (d) 1.67CPC promoted RANKL-induced phosphorylation of NF-κB p65, and degradation of IκBα. (e) p-p65 and (f) IκBα intensity of Western blot bands was calculated with normalizing to β-actin. The untreated medium was treated as control. (g–l) Relative mRNA expression of osteoclast was measured after inducing BMMs for 7 days in CPC extract. BMMs cultured with untreated medium was treated as control. (*P ＜ 0.05, **P ＜ 0.01,***P ＜ 0.001).

Article Snippet: The concentration of RANK was measured by Mouse RANK ELISA Kit (Boster Biological Technology, China), according to the manufacturer’s instructions and the total protein concentration was measured by BCA Protein Assay Kit (Beyotime, China), which was used to normalize the expression of RANK.

Techniques: Immunofluorescence, Expressing, Concentration Assay, Enzyme-linked Immunosorbent Assay, Phospho-proteomics, Western Blot, Control, Cell Culture

Journal: The Journal of Biological Chemistry

Article Title: IL-1β-induced and p38 MAPK -dependent activation of the mitogen-activated protein kinase-activated protein kinase 2 (MK2) in hepatocytes: Signal transduction with robust and concentration-independent signal amplification

doi: 10.1074/jbc.M117.775023

Figure Lengend Snippet: Quantification of p38MAPK phosphorylation in primary mouse hepatocytes. A, immunoblotting of whole cell lysates in co-separation with an internal standard affords the quantification of intracellular phosphorylated p38MAPK molecule concentration. Primary mouse hepatocytes were treated with 0, 0.1, 0.5, and 1 ng·ml−1 IL-1β, respectively, for up to 60 min. The cells lysed in Triton lysis buffer and specimen were applied randomly to SDS-PAGE and subsequent Western blotting in co-separation with a dilution series (1, 5, 10, 20, and 40 μl) of an internal standard (int). After incubation with specific antibodies, the nitrocellulose membrane was cut in half to fit on the documentation screen, and chemiluminescence was captured at a 16-bit charge-coupled device. B and C, quantitative determination of IL-1β-induced p38MAPK phosphorylation kinetics. The intensity of the chemiluminescent signals was quantified, and, in consideration of cell volume and the internal standard, the intracellular concentration of p38MAPK phosphorylation as a function of IL-1β and time was quantified. a. u.: arbitrary units.

Article Snippet: In vitro phosphorylation of recombinant proteins Purified recombinant GST-p38 MAPK was incubated for 30 min in the presence of constitutively active human MKK6 (Sigma), whereas, for in vitro phosphorylation of recombinant GST-MK2, constitutively active human p38 MAPK (R&D Systems, Minneapolis, MN) was used.

Techniques: Western Blot, Concentration Assay, Lysis, SDS Page, Incubation

Journal: The Journal of Biological Chemistry

Article Title: IL-1β-induced and p38 MAPK -dependent activation of the mitogen-activated protein kinase-activated protein kinase 2 (MK2) in hepatocytes: Signal transduction with robust and concentration-independent signal amplification

doi: 10.1074/jbc.M117.775023

Figure Lengend Snippet: ODE model of IL-1β-dependent p38MAPK/MK2 activation. A, mathematical model of IL-1β-dependent p38MAPK/MK2 activation. Shown is a graphical representation of the ODE model of IL-1β-dependent p38MAPK and MK2 signal transduction. R1, basal and IL-1β-induced activation (Thr-180/Tyr-182 phosphorylation) of p38MAPK; R2, dephosphorylation of p38MAPK by MKP; R3, activation (Thr-222 phosphorylation) of MK2 by activated p38MAPK; R4, dephosphorylation of MK2; R5, activated p38MAPK induces the expression of MKP; R6, degradation of MKP. The reaction rates of the system are forming a five-dimensional ODE.

Article Snippet: In vitro phosphorylation of recombinant proteins Purified recombinant GST-p38 MAPK was incubated for 30 min in the presence of constitutively active human MKK6 (Sigma), whereas, for in vitro phosphorylation of recombinant GST-MK2, constitutively active human p38 MAPK (R&D Systems, Minneapolis, MN) was used.

Techniques: Activation Assay, Transduction, De-Phosphorylation Assay, Expressing

Journal: The Journal of Biological Chemistry

Article Title: IL-1β-induced and p38 MAPK -dependent activation of the mitogen-activated protein kinase-activated protein kinase 2 (MK2) in hepatocytes: Signal transduction with robust and concentration-independent signal amplification

doi: 10.1074/jbc.M117.775023

Figure Lengend Snippet: Quantitative determination of IL-1β-induced p38MAPK and MK2 phosphorylation kinetics. Primary mouse hepatocytes were treated with 0–40 ng·ml−1 IL-1β for up to 60 min. Whole cell lysates were applied to Western blotting analysis with a serial dilution of an internal standard, and phosphorylation of p38MAPK at Thr-180/Tyr-182 as well as phosphorylation of MK2 at Thr-222 was detected by respective phosphorylation-specific antibodies. Signal intensities were determined and converted to intracellular concentrations. The solid lines indicate the fit to the mathematical model based on ordinary differential equations, whereas symbols indicate the respective experimental data. In total, the experimental data used for model fitting are from 18 independent experiments (biological replicates). Each experiment was done in up to three technical replicates to account for measuring inaccuracy.

Article Snippet: In vitro phosphorylation of recombinant proteins Purified recombinant GST-p38 MAPK was incubated for 30 min in the presence of constitutively active human MKK6 (Sigma), whereas, for in vitro phosphorylation of recombinant GST-MK2, constitutively active human p38 MAPK (R&D Systems, Minneapolis, MN) was used.

Techniques: Western Blot, Serial Dilution

Journal: The Journal of Biological Chemistry

Article Title: IL-1β-induced and p38 MAPK -dependent activation of the mitogen-activated protein kinase-activated protein kinase 2 (MK2) in hepatocytes: Signal transduction with robust and concentration-independent signal amplification

doi: 10.1074/jbc.M117.775023

Figure Lengend Snippet: Simulation of the p38MAPK and MK2 phosphorylation kinetics as a function of IL-1β concentration and time. A and B, simulations based on the fitted model illustrate the time- and dose-dependent kinetics of IL-1β-dependent p38MAPK (A) and MK2 (B) activation, where the intrinsic signal amplification is reflected. Signal amplification is roughly IL-1β concentration (conc)-independent, and signal transduction occurs under minimal deformation.

Article Snippet: In vitro phosphorylation of recombinant proteins Purified recombinant GST-p38 MAPK was incubated for 30 min in the presence of constitutively active human MKK6 (Sigma), whereas, for in vitro phosphorylation of recombinant GST-MK2, constitutively active human p38 MAPK (R&D Systems, Minneapolis, MN) was used.

Techniques: Concentration Assay, Activation Assay, Amplification, Transduction

Journal: The Journal of Biological Chemistry

Article Title: IL-1β-induced and p38 MAPK -dependent activation of the mitogen-activated protein kinase-activated protein kinase 2 (MK2) in hepatocytes: Signal transduction with robust and concentration-independent signal amplification

doi: 10.1074/jbc.M117.775023

Figure Lengend Snippet: Parameter likelihood profiles. Likelihood profiles of the dynamic model parameters (dePhospho_inh, il1b_sat, init_MK2, init_p38, mk2_act_p38, mk2_dea, mk2_inh_sb, mkp_prod_pp38, p38_act_basal, p38_act_il1b, p38_dea_const, and p38_dea_mkp). Black lines represent the profile likelihood. The parameter set of the optimum is indicated with a green dot. Red lines indicate the 95% confidence level. Blue lines indicate the global minimum of the parameter estimation process. The analysis revealed that all model parameters are structurally identifiable. The parameter p38_dea_const is practically non-identifiable. The parameter profile suggests that the parameter is compatible with zero; i.e. the constant dephosphorylation rate of p38MAPK is not relevant for our system.

Article Snippet: In vitro phosphorylation of recombinant proteins Purified recombinant GST-p38 MAPK was incubated for 30 min in the presence of constitutively active human MKK6 (Sigma), whereas, for in vitro phosphorylation of recombinant GST-MK2, constitutively active human p38 MAPK (R&D Systems, Minneapolis, MN) was used.

Techniques: De-Phosphorylation Assay

Journal: The Journal of Biological Chemistry

Article Title: IL-1β-induced and p38 MAPK -dependent activation of the mitogen-activated protein kinase-activated protein kinase 2 (MK2) in hepatocytes: Signal transduction with robust and concentration-independent signal amplification

doi: 10.1074/jbc.M117.775023

Figure Lengend Snippet: In silico analysis (predictions) and experimental validation. A and C, contribution of kinases and phosphatases to signal amplitude and duration. Simulations of parameter perturbations that are reflecting changes in kinase and phosphatase activity are showing different effects on MK2 phosphorylation. The perturbation of the kinases was simulated by changing the parameter mk2_act_p38 by a factor indicated in the figure. This has mainly an effect on the signal amplitude of MK2 phosphorylation. The perturbation of the phosphatases was modeled by changing the parameter p38_dea_MKP. This affects both signal amplitude and duration of MK2 phosphorylation. B, SB203580 inhibited IL-1β induced MK2 phosphorylation. Primary mouse hepatocytes were treated with the p38MAPK inhibitor SB203580 for 30 min prior to treatment with 1 ng·ml−1 IL-1β. Whole cell lysates were applied to Western blotting analysis with a serial dilution of an internal phosphostandard, and phosphorylation of MK2 at Thr-222 was detected by specific antibodies. Signal intensities were determined and converted to intracellular concentrations. The data were fitted to the mathematical model (solid lines). D, inhibition of p38MAPK and MK2 dephosphorylation. Primary mouse hepatocytes were treated with 200 μm sodium orthovanadate and 200 μm β-glycerophosphate for inhibition of phosphatases prior to treatment with 1 ng·ml−1 IL-1β. Whole cell lysates were applied to Western blotting analysis, phosphorylation of MK2 at Thr-222 (B) was detected by specific antibodies, and the signal intensities were determined. The data were fitted to the mathematical model (solid lines).

Article Snippet: In vitro phosphorylation of recombinant proteins Purified recombinant GST-p38 MAPK was incubated for 30 min in the presence of constitutively active human MKK6 (Sigma), whereas, for in vitro phosphorylation of recombinant GST-MK2, constitutively active human p38 MAPK (R&D Systems, Minneapolis, MN) was used.

Techniques: In Silico, Activity Assay, Western Blot, Serial Dilution, Inhibition, De-Phosphorylation Assay

Journal: The Journal of Biological Chemistry

Article Title: IL-1β-induced and p38 MAPK -dependent activation of the mitogen-activated protein kinase-activated protein kinase 2 (MK2) in hepatocytes: Signal transduction with robust and concentration-independent signal amplification

doi: 10.1074/jbc.M117.775023

Figure Lengend Snippet: Comparative analyses between hepatocytes and macrophages. A, absolute concentrations. The concentrations of total and phosphorylated p38MAPK and MK2 were measured with the internal standard for different durations and concentrations of IL-1β treatment of macrophages (red) and hepatocytes (gray), respectively. The total concentrations are significantly higher in macrophages in comparison with hepatocytes. The criteria for significance were a two-way analysis of variance and post hoc pairwise comparisons of each IL-1β treatment condition of hepatocytes and macrophages, respectively, based on multiple two-tailed t tests. The p values were adjusted according to Bonferroni to account for the multiple comparisons problem. ****, p < 0.0001. B, kinetics. The kinetics of phosphorylated MK2 and p38MAPK were measured in macrophages and hepatocytes for different IL-1β inputs (points). For hepatocytes, the solid lines indicate the prediction of the calibrated model. For macrophages, the mathematical model was fitted to the measured macrophage data (solid line). C, model prediction of dose responses. Shown is relative phosphorylation dependent on the IL-1β input dose as predicted with the calibrated models. The bands show the prediction uncertainty calculated by integrating all parameter sets along the corresponding profile likelihoods.

Article Snippet: In vitro phosphorylation of recombinant proteins Purified recombinant GST-p38 MAPK was incubated for 30 min in the presence of constitutively active human MKK6 (Sigma), whereas, for in vitro phosphorylation of recombinant GST-MK2, constitutively active human p38 MAPK (R&D Systems, Minneapolis, MN) was used.

Techniques: Two Tailed Test

Journal: The Journal of Biological Chemistry

Article Title: IL-1β-induced and p38 MAPK -dependent activation of the mitogen-activated protein kinase-activated protein kinase 2 (MK2) in hepatocytes: Signal transduction with robust and concentration-independent signal amplification

doi: 10.1074/jbc.M117.775023

Figure Lengend Snippet: Schematic of the signal amplification within the IL-1β-induced p38MAPK and MK2 pathway in hepatocytes. Under basal conditions, only a small fraction of p38MAPK and MK2 is activated, i.e. 1.3 nm of 359 nm and 85.6 nm of 4637 nm, respectively. After treatment with up to 2.3 nm IL-1β, under saturating conditions, the concentration of phosphorylated p38MAPK and MK2 rises transiently to 40.6 and 1693 nm. Under subsaturated conditions, e.g. 57 pm, IL-1β induces an activation of approximately 21 nm p38MAPK via the IL1 receptor complex. This subsequently induces an activation of 1063 nm MK2.

Article Snippet: In vitro phosphorylation of recombinant proteins Purified recombinant GST-p38 MAPK was incubated for 30 min in the presence of constitutively active human MKK6 (Sigma), whereas, for in vitro phosphorylation of recombinant GST-MK2, constitutively active human p38 MAPK (R&D Systems, Minneapolis, MN) was used.

Techniques: Amplification, Concentration Assay, Activation Assay