Journal: Journal of Cellular and Molecular Medicine

Article Title: Quercetin inhibits macrophage polarization through the p‐38α/β signalling pathway and regulates OPG/RANKL balance in a mouse skull model

doi: 10.1111/jcmm.14995

Figure Lengend Snippet: A, The effect of quercetin on p‐p38 by immunofluorescence. B, Ti wear particles activated high expression of p‐p38. C, The inhibitor of p38 and quercetin inhibited the activation of p‐p38 by Ti wear particles. D‐G showed that quercetin, p38 inhibitor, Si‐p38α RNA and Si‐p38β RNA significantly inhibited M1 macrophages from promoting M2 macrophages

Article Snippet: The antibodies (GAPDH, NFkB, C‐FOS, NFATc1, p‐p38,) were purchased from Cell Signaling Technology, Inc. Enzyme‐linked immunosorbent assay (ELISA) kits (IL‐6, IL‐1β, TNF‐α, IL‐10, Arg‐1, iNOS) were purchased from R&D Systems, Inc Flow cytometry anti‐mouse CD16/32‐PE (cat. no.553145) and anti‐mouse CD206‐Alexa 647 (cat. no.565250) were purchased from BioLegend Inc The p38α/β MAPK inhibitor (SB202190) was purchased from Selleck.

Techniques: Immunofluorescence, Expressing, Activation Assay

Journal: Scientific Reports

Article Title: Neuronal MAP kinase p38α inhibits c-Jun N-terminal kinase to modulate anxiety-related behaviour

doi: 10.1038/s41598-018-32592-y

Figure Lengend Snippet: Mice with neuron-specific p38α deletion show abnormal anxiety-related responses. ( A – D ) Anxiety-related response in mice was tested for 5 minutes using the elevated plus maze (EPM) with p38α lox/lox and p38α ΔNeu mice. (n = 10–11) ( A ) Representative EPM traces. Traces reflect movement within the first minute of testing. OA, open arm; CA, closed arm. ( B ) Time in the open arms ( C ) Time in the closed arms. ( D ) Ratio of time in open/time in closed arms. ( E – H ) Activity in a novel environment was addressed using the open field paradigm (OFT) with p38α lox/lox and p38α ΔNeu mice. (n = 10–16) ( E ) Representative OFT traces. ( F ) Average speed in OFT ( G ) total distance covered in OFT. ( H ) Thigmotaxis index in OFT ( I ) Object recognition memory was addressed using the novel object recognition test (NOR) with p38α lox/lox and p38α ΔNeu mice. (n = 10–16) Ratio of time spent with novel object/time spent with familiar object is shown. ( J – N ) Spatio-temporal memory acquisition and retrieval was tested using the Morris water maze (MWM) with p38α lox/lox and p38α ΔNeu mice. (n = 8–10) ( J ) Representative MWM traces on acquisition day 6 ( K ) MWM acquisition curve on days 1–6 ( L ) MWM quadrant occupancy during probe trial on day 7. ( M ) Escape latency during visual cued trial on day 8 ( N ) Average swim speed during probe trial on day 7. ( O ) Motor assessment using the Rotarod with p38α lox/lox and p38α ΔNeu mice. (n = 13–14) Average latency to fall is shown. Values are mean ± S.E.M. (Student’s t-test) *** p < 0.001, * p < 0.05 ns, non-significant.

Article Snippet: Antibodies used were phospho-T202/Y204 ERK (D13.14.4E; Cell Signaling Technologies), ERK (Cell Signaling Technologies), phospho-T183/Y185 JNK (81E11; Cell Signaling Technologies), phospho-T183/Y185 JNK (98F2; Cell Signaling Technologies), JNK1/2/3 (Cell Signaling Technologies), JNK (Sigma), phospho-T180/Y182 p38 (D3F9; Cell Signaling Technologies), p38α (Cell Signaling Technologies), GAPDH (Millipore), NeuN (Abcam), GFAP (Abcam).

Techniques: Activity Assay

Journal: Scientific Reports

Article Title: Neuronal MAP kinase p38α inhibits c-Jun N-terminal kinase to modulate anxiety-related behaviour

doi: 10.1038/s41598-018-32592-y

Figure Lengend Snippet: p38α impairs activation of JNK. ( A ) Expression of active p38α inhibits stimulated JNK activation in cultured cells. 293 T cells were transfected with constructs expressing either enhanced green fluorescent protein (eGFP) as control or constitutively active p38α (p38α CA ). After stimulation with anisomycin (ANI; 25 ng/ml for 30 minutes) or vehicle (VEH), cell lysates were prepared for immunoblots for pThr183/pTyr185 JNK (pJNK), JNK (Cell Signaling Technologies), pThr180/Tyr182 p38 (p-p38) and p38α. GAPDH, loading control. ( B ) Quantification of immunoblots from three independent experiments (Student’s t-test) *** p < 0.001 ( C ) CRISPR/Cas9-mediated ablation of p38α increases stimulated JNK activation. eGFP-sorted 293 T cells transfected with Cas9 and p38α gRNA-expressing constructs (CRISPR p38α ) or Cas9-expressing empty vector (Cas9 EV). Two different p38α gRNAs were co-transfected. After stimulation with anisomycin (ANI; 25 ng/ml for 30 minutes) or vehicle (VEH), cell lysates were prepared for immunoblots for pThr183/pTyr185 JNK (pJNK), JNK (Sigma) and p38α. GAPDH, loading control. ( D ) Quantification of immunoblots from three independent experiments (Student’s t-test) *** p < 0.001 ( F ) Of the four p38 MAPKs, only p38α inhibits JNK activation. 293 T cells were transfected with constructs expressing constitutively active hemagglutinin (HA)-tagged p38α (p38α CA ), HA-tagged p38β (p38β CA ), HA-tagged p38γ (p38γ CA ) or p38δ (p38α CA ). Cells expressing eGFP served as control. After stimulation with anisomycin (ANI; 25 ng/ml for 30 minutes) or vehicle (VEH), cell lysates were prepared for immunoblots for pThr183/pTyr185 JNK (pJNK), JNK, pThr180/Tyr182 p38 (p-p38), HA and p38δ. GAPDH, loading control. Representative blots from two experiments are shown.

Article Snippet: Antibodies used were phospho-T202/Y204 ERK (D13.14.4E; Cell Signaling Technologies), ERK (Cell Signaling Technologies), phospho-T183/Y185 JNK (81E11; Cell Signaling Technologies), phospho-T183/Y185 JNK (98F2; Cell Signaling Technologies), JNK1/2/3 (Cell Signaling Technologies), JNK (Sigma), phospho-T180/Y182 p38 (D3F9; Cell Signaling Technologies), p38α (Cell Signaling Technologies), GAPDH (Millipore), NeuN (Abcam), GFAP (Abcam).

Techniques: Activation Assay, Expressing, Cell Culture, Transfection, Construct, Control, Western Blot, CRISPR, Plasmid Preparation

Journal: Scientific Reports

Article Title: Neuronal MAP kinase p38α inhibits c-Jun N-terminal kinase to modulate anxiety-related behaviour

doi: 10.1038/s41598-018-32592-y

Figure Lengend Snippet: Neuron-specific p38α deletion results in increased hippocampal JNK activation in mice. ( A ) Immunoblots of hippocampal lysates (RIPA buffer with 10 µM okadaic acid) from p38α lox/lox and p38α ΔNeu mice (n = 5) were probed for pThr183/pTyr185 JNK (pJNK) using 2 independent antibodies (pJNK Ab#1 and Ab#2), JNK, pThr202/pTyr204 ERK (pERK), ERK and p38α. Gapdh, loading control. Note the high levels of pJNK in p38α ΔNeu as compared to p38α lox/lox . ( B ) Quantification of immunoblots shown in ( A ). p38α levels are expressed relative to Gapdh levels. pJNK and pERK levels are expressed relative to total JNK and total ERK, respectively, and normalized to Gapdh levels. (n = 5) values are mean ± S.E.M. (Student’s t-test) *** p < 0.001; ns, non-significant. ( C ) Hippocampal sections (5 µm) of p38α lox/lox and p38α ΔNeu mice were prepared and immunostained for astrocytic marker GFAP and neuronal nuclear marker NeuN. Scale bar, 100 µm. Note the similar hippocampal structure, neuronal and astrocytic numbers in p38α lox/lox and p38α ΔNeu mice. ( D ) Thickness of NeuN + cell layers in the hippocampus of p38α lox/lox and p38α ΔNeu mice. CA1, cornu ammonis 1; CA3, cornu ammonis 3; DG, dentate gyrus. (n = 5) values are mean ± S.E.M. (Student’s t-test) ns, non-significant. ( E ) Numbers of GFAP-positive astrocytic cells in the hippocampus of p38α lox/lox and p38α ΔNeu mice. MOL, molecular layer; SR, stratum radiatum. (n = 5) values are mean ± S.E.M. (Student’s t-test) ns, non-significant.

Article Snippet: Antibodies used were phospho-T202/Y204 ERK (D13.14.4E; Cell Signaling Technologies), ERK (Cell Signaling Technologies), phospho-T183/Y185 JNK (81E11; Cell Signaling Technologies), phospho-T183/Y185 JNK (98F2; Cell Signaling Technologies), JNK1/2/3 (Cell Signaling Technologies), JNK (Sigma), phospho-T180/Y182 p38 (D3F9; Cell Signaling Technologies), p38α (Cell Signaling Technologies), GAPDH (Millipore), NeuN (Abcam), GFAP (Abcam).

Techniques: Activation Assay, Western Blot, Control, Marker

Journal: Scientific Reports

Article Title: Neuronal MAP kinase p38α inhibits c-Jun N-terminal kinase to modulate anxiety-related behaviour

doi: 10.1038/s41598-018-32592-y

Figure Lengend Snippet: D-JNKi reduces JNK activation in neuronal p38α knockout mice to levels in control mice. ( A ) 293 T cells were treated with D-JNKi (1 μM), SP600125 (10 μM) for 30 minutes at 37 °C. After stimulation with anisomycin (ANI; 25 ng/ml for 30 minutes) or vehicle (VEH), cell lysates were prepared for immunoblots for pThr183/pTyr185 JNK (pJNK), JNK. GAPDH, loading control. ( B ) Quantification of immunoblots in ( A ). (n = 3) (Student’s t-test) *** p < 0.001. ( C ) Immunoblots of hippocampal lysates from p38α lox/lox and p38α ΔNeu mice 30 minutes post-application (i.p.) of D-JNKi (0.3 mg/kg body weight) or control vehicle (VEH). Immunoblots were probed for pThr183/pTyr185 JNK (pJNK), JNK, p38α and Gapdh. 30 minutes post injection of D-JNKi, activation levels of JNK were comparable between p38α lox/lox and p38α ΔNeu brains, whereas control-treated p38α ΔNeu brains show consistently higher levels of active JNK than control-treated p38α lox/lox . (n = 5) ( D ) Quantification of immunoblots in (A). (n = 5) (Student’s t-test) *** p < 0.001.

Article Snippet: Antibodies used were phospho-T202/Y204 ERK (D13.14.4E; Cell Signaling Technologies), ERK (Cell Signaling Technologies), phospho-T183/Y185 JNK (81E11; Cell Signaling Technologies), phospho-T183/Y185 JNK (98F2; Cell Signaling Technologies), JNK1/2/3 (Cell Signaling Technologies), JNK (Sigma), phospho-T180/Y182 p38 (D3F9; Cell Signaling Technologies), p38α (Cell Signaling Technologies), GAPDH (Millipore), NeuN (Abcam), GFAP (Abcam).

Techniques: Activation Assay, Knock-Out, Control, Western Blot, Injection

Journal: Scientific Reports

Article Title: Neuronal MAP kinase p38α inhibits c-Jun N-terminal kinase to modulate anxiety-related behaviour

doi: 10.1038/s41598-018-32592-y

Figure Lengend Snippet: Inhibition of JNK restores anxiety-related behaviour in neuronal p38α knockout mice. ( A – D ) EPM tests (2 minutes) in p38α lox/lox and p38α ΔNeu mice 30 minutes post-application (i.p.) of D-JNKi (0.3 mg/kg body weight) or control vehicle (VEH). (n = 15–16). ( A ) Representative EPM traces reflect movement within the first 2 minutes of testing. OA, open arm; CA, closed arm. ( B ) Time in the open arms. (C) Time in the closed arms. ( D ) Ratio of time in open/time in closed arms. Values are mean ± S.E.M. (ANOVA) ** p < 0.01, * p < 0.05 ns, non-significant.

Article Snippet: Antibodies used were phospho-T202/Y204 ERK (D13.14.4E; Cell Signaling Technologies), ERK (Cell Signaling Technologies), phospho-T183/Y185 JNK (81E11; Cell Signaling Technologies), phospho-T183/Y185 JNK (98F2; Cell Signaling Technologies), JNK1/2/3 (Cell Signaling Technologies), JNK (Sigma), phospho-T180/Y182 p38 (D3F9; Cell Signaling Technologies), p38α (Cell Signaling Technologies), GAPDH (Millipore), NeuN (Abcam), GFAP (Abcam).

Techniques: Inhibition, Knock-Out, Control

Journal: Plants

Article Title: In Vitro Anti-Inflammatory Effects of Symplocos sumuntia Buch.-Ham. Ex D. Don Extract via Blockage of the NF-κB/JNK Signaling Pathways in LPS-Activated Microglial Cells

doi: 10.3390/plants11223095

Figure Lengend Snippet: Effects of the SSME on the MAPK signaling pathway. The BV2 cells were treated with SSME (50, 100, and 200 µg/mL) for 2 h, followed by stimulation with LPS (1 µg/mL) for 15 min. The cellular protein levels of p38, p44/42 (Erk1/2), and JNK were measured by western blotting. The data are presented as the mean ± SEM of three independent experiments. Differences between the groups were analyzed using the Mann–Whitney U test. # p < 0.05, compared with the untreated group and the LPS-treated groups; * p < 0.05, compared with the LPS-treated and SSME- and LPS-treated groups. p-, phosphorylated; SAPK/JNK, stress-associated protein kinase/c-Jun N-terminal kinase; ns, not significant.

Article Snippet: The membranes were incubated with anti-iNOS (1:1000; #610332; BD Biosciences, San Diego, CA, USA), anti-COX2 (1:1000; #sc-166475), anti-β-actin (1:5000; #sc-47778), anti-p38 (1:2000; #sc-7972), anti-p44/42 (1:2000; Erk1/2, #sc-514302) (Santa Cruz Biotechnology Inc., Dallas, TX, USA), anti-SAPK/JNK (1:1000; #9252), anti-phospho-SAPK/JNK (1:1000; #9251), anti-phospho-p38 (1:1000; #9211), and anti-phospho-p44/42 (1:2000; #9101) (Cell Signaling Technology, Danvers, MA, USA) overnight at 4 °C.

Techniques: Western Blot, MANN-WHITNEY

Journal: bioRxiv

Article Title: Dynamic and structural insights into allosteric regulation on MKP5 a dual-specificity phosphatase

doi: 10.1101/2024.09.05.611520

Figure Lengend Snippet: Full-length GST-tagged MKP5 WT, Y435 mutants, and C2408S were transiently transfected with p38α MAPK-HA, JNK1-β-GFP, or ERK2-HA into COS-7 cells for 48 h. Cells were harvested and lysed, followed by affinity purification of GSH Sepharose beads. (a) GST affinity complexes and whole cell lysates were immunoblotted with anti-HA and anti-GST antibodies. (b) GST affinity complexes and whole cell lysates were immunoblotted with anti-GFP and anti-GST antibodies. Graphs shown below represent quantitation of a , p38α MAPK-HA/GST-MKP5 and b , JNK1-GFP/GST-MKP5. Data represent the mean ± SEM of three to four to five independent experiments. Statistical significance shown was generated using a two-way ANOVA. Key: *; p <0.05, **; p <0.01, ***; p <0.005 and p <0.0001.

Article Snippet: Total levels of MAPKs were assessed by immunoblotting of re-probed membranes using anti-p38α MAPK (Santa Cruz cat# 81621), anti-JNK1/2 (Santa Cruz cat# 3708), and anti-ERK1/2 (Cell Signal cat# 9107) antibodies.

Techniques: Transfection, Affinity Purification, Quantitation Assay, Generated

Journal: bioRxiv

Article Title: Dynamic and structural insights into allosteric regulation on MKP5 a dual-specificity phosphatase

doi: 10.1101/2024.09.05.611520

Figure Lengend Snippet: Full-length GST-tagged MKP5 WT, Y435A, C408S and C435/C408S mutants were transiently transfected with p38α MAPK-HA, JNK1-β-GFP into COS-7 cells for 48 h. Cells were harvested and lysed, followed by affinity purification with GSH Sepharose beads. (a) GST affinity complexes and whole cell lysates were immunoblotted with anti-HA, anti-GST and phospho-p38 MAPK antibodies. (b) Densitometric quantitation of relative normalized p38α MAPK bound to GST-MKP5; (c) Relative normalized phosphorylated p38 MAPK in complex with MKP5; (d) GFP affinity complexes and whole cell lysates were immunoblotted with anti-GFP, anti-GST and phospho-JNK antibodies. (e) Graph showing densitometric quantitation of relative normalized JNK bound to GST-MKP5; (f) Graph showing relative normalized phosphorylated JNK in complex with GST-MKP5. Data represent the mean ± SEM of four independent experiments. Statistical significance shown was generated using a two-way ANOVA. Key, *; p<0.05

Article Snippet: Total levels of MAPKs were assessed by immunoblotting of re-probed membranes using anti-p38α MAPK (Santa Cruz cat# 81621), anti-JNK1/2 (Santa Cruz cat# 3708), and anti-ERK1/2 (Cell Signal cat# 9107) antibodies.

Techniques: Transfection, Affinity Purification, Quantitation Assay, Generated