Journal: Neural Regeneration Research

Article Title: Thrombin increases the expression of cholesterol 25-hydroxylase in rat astrocytes after spinal cord injury

doi: 10.4103/1673-5374.357905

Figure Lengend Snippet: Effects of thrombin/protease-activated receptor 1 on the mitogen-activated protein kinase (MAPK)/nuclear factor kappa-B (NFκB) pathway in astrocytes. (A) Western blot analysis of phosphorylated extracellular regulated protein kinase (ERK), c-Jun N-terminal kinase (JNK), P38 kinase (P38), and NFκB protein levels following astrocyte stimulation with 0, 0.5, 1, or 2 U/mL thrombin for 24 hours. (B–E) Quantification of phosphorylated ERK, JNK, P38, and NFκB protein levels, normalized to β-actin. (F) Western blot analysis of phosphorylated ERK, JNK, P38, and NFκB protein levels following astrocyte stimulation with 1 U/mL thrombin in the presence of 0–3 μM SCH79797 for 24 hours. (G–J) Quantification of phosphorylated ERK, JNK, P38, and NFκB protein levels, normalized to β-actin. Data are expressed as mean ± SEM ( n = 3). * P < 0.05 (one-way analysis of variance followed by Bonferroni’s post hoc comparison test). SCH79797: Protease-activated receptor 1 inhibitor.

Article Snippet: After blocking with 5% skim milk for 1 hour, the membrane was incubated with the following primary antibodies overnight at 4°C: CH25H (rabbit, 1:500, Invitrogen, Cat# PA5-70691, RRID: AB_2689560), phosphorylated extracellular signal regulated kinase (pERK)1/2 (rabbit, 1:1000, CST, Cat# 9102S, RRID: AB_330744), extracellular signal regulated kinase (ERK)1/2 (rabbit, 1:1000, CST, Cat# 8544S, RRID: AB_11127856), phosphorylated c-Jun N-terminal kinase (pJNK; rabbit, 1:1000, CST, Cat# 9251S, RRID: AB_331659), JNK (rabbit, 1:1000, CST, Cat# 9252S, RRID: AB_2250373), phosphorylated P38 kinase (pP38; rabbit, 1:1000, CST, Cat# 8632S, RRID: AB_2797648), P38 (rabbit, 1:1000, CST, Cat# 14451S, RRID: AB_2798482), nuclear factor kappa-B (NFκB; rabbit, 1:1000, CST, Cat# 12629S, RRID: AB_2722509), and β-actin (mouse, 1:5000, CST, Cat# 3700S, RRID: AB_2242334).

Techniques: Western Blot

Journal: Neural Regeneration Research

Article Title: Thrombin increases the expression of cholesterol 25-hydroxylase in rat astrocytes after spinal cord injury

doi: 10.4103/1673-5374.357905

Figure Lengend Snippet: Effects of mitogen-activated protein kinase (MAPK) or nuclear factor kappa-B (NFκB) inhibitors on astrocyte cholesterol-25-hydroxylase (CH25H) expression in response to stimulation with thrombin. (A) Western blot analysis of NFκB expression following treatment with 1 U/mL thrombin in the presence of 10 μM PD98059, 10 μM SP600125, or 10 μM SB203580 for 24 hours. (B) Quantification of NFκB protein expression, normalized to β-actin. (C) Western blot analysis of CH25H expression following treatment with 1 U/mL thrombin in the presence of 10 μM PD98059, 10 μM SP600125, or 10 μM SB203580 for 24 hours. (D) Quantification of CH25H expression, normalized to β-actin. (E) Western blot analysis of CH25H expression following treatment with 1 U/mL thrombin in the presence or absence of 10–100 μM of the NFκB inhibitor PDTC for 24 hours. (F) Quantification of CH25H expression, normalized to β-actin. Data are expressed as mean ± SEM ( n = 3). * P < 0.05 (one-way analysis of variance followed by Bonferroni’s post hoc comparison test). DMSO: Dimethyl sulfoxide; PD98059: extracellular regulated protein kinases inhibitor; SB203580: P38 inhibitor; SP600125: c-Jun N-terminal kinase inhibitor.

Article Snippet: After blocking with 5% skim milk for 1 hour, the membrane was incubated with the following primary antibodies overnight at 4°C: CH25H (rabbit, 1:500, Invitrogen, Cat# PA5-70691, RRID: AB_2689560), phosphorylated extracellular signal regulated kinase (pERK)1/2 (rabbit, 1:1000, CST, Cat# 9102S, RRID: AB_330744), extracellular signal regulated kinase (ERK)1/2 (rabbit, 1:1000, CST, Cat# 8544S, RRID: AB_11127856), phosphorylated c-Jun N-terminal kinase (pJNK; rabbit, 1:1000, CST, Cat# 9251S, RRID: AB_331659), JNK (rabbit, 1:1000, CST, Cat# 9252S, RRID: AB_2250373), phosphorylated P38 kinase (pP38; rabbit, 1:1000, CST, Cat# 8632S, RRID: AB_2797648), P38 (rabbit, 1:1000, CST, Cat# 14451S, RRID: AB_2798482), nuclear factor kappa-B (NFκB; rabbit, 1:1000, CST, Cat# 12629S, RRID: AB_2722509), and β-actin (mouse, 1:5000, CST, Cat# 3700S, RRID: AB_2242334).

Techniques: Expressing, Western Blot

Journal: Journal of Cellular and Molecular Medicine

Article Title: Hsa‐miR ‐409‐3p regulates endothelial progenitor senescence via PP2A‐P38 and is a potential ageing marker in humans

doi: 10.1111/jcmm.17691

Figure Lengend Snippet: Angiogenic activities of young human EPCs after treatment with siRNA specific to PP2A (siPP2A) and western blot analysis of young human EPCs treated with PP2A siRNA. Note that (A) transcript and (B and C) protein expression levels of PP2A were suppressed in siPP2A‐treated EPCs, accompanied by attenuated cellular activities of (D) proliferation, (E) migration and (F) tube formation. n = 3 for each bar. (G) In three different clones of EPCs, phosphorylated p38 (p‐p38) and phosphorylated JNK (p‐JNK) were upregulated after siPP2A treatment. Quantitative Analysis confirmed the activation of (H) p38 and (I) JNK signalling pathways. n = 3 for each bar. Data are mean ± SEM and analysed using unpaired Student's t test for comparison between groups. * p < 0.05, *** p < 0.001 vs. corresponding negative control (NC). Abbreviations are as in Figure

Article Snippet: The antibodies included phosphorylated p38 mitogen‐activated protein kinase (p‐p38), total p38, phosphorylated c‐Jun N‐terminal kinase (p‐JNK), total JNK, phosphorylated extracellular signal‐regulated kinase (p‐ERK) and total ERK from Cell Signalling Technology.

Techniques: Western Blot, Expressing, Migration, Clone Assay, Activation Assay, Negative Control

Journal: Journal of Cellular and Molecular Medicine

Article Title: Hsa‐miR ‐409‐3p regulates endothelial progenitor senescence via PP2A‐P38 and is a potential ageing marker in humans

doi: 10.1111/jcmm.17691

Figure Lengend Snippet: Regulation of PP2A by miR‐409‐3p and the signalling proteins in young human EPCs. PP2A was downregulated by miR‐409‐3p mimic at (A) transcript and (B and C) protein levels, compared to corresponding NC. (D) Time series analysis showed, in cells treated with the mimic, (E) PP2A was downregulated for at least 72 h, while (F) p‐p38 and (G) p‐JNK were activated with peaks at 48 h. n = 5 for each bar. Data are mean ± SEM and analysed using one‐way anova followed by post hoc Fisher's least significant difference test for multiple group comparisons. * p < 0.05 vs. NC. Abbreviations are as in Figure

Article Snippet: The antibodies included phosphorylated p38 mitogen‐activated protein kinase (p‐p38), total p38, phosphorylated c‐Jun N‐terminal kinase (p‐JNK), total JNK, phosphorylated extracellular signal‐regulated kinase (p‐ERK) and total ERK from Cell Signalling Technology.

Techniques:

Journal: Journal of Cellular and Molecular Medicine

Article Title: Hsa‐miR ‐409‐3p regulates endothelial progenitor senescence via PP2A‐P38 and is a potential ageing marker in humans

doi: 10.1111/jcmm.17691

Figure Lengend Snippet: Prediction of binding site of hsa‐miR‐409‐3p for protein phosphatase 2 catalytic subunit alpha (PPP2CA) gene and examination of its product protein phosphatase 2A (PP2A). (A) Diagram showing the seed sequences of hsa‐miR‐409‐3p and its predicted binding site in the 3’‐UTR of PPP2CA gene by prediction methods including TargetScan and miRanda. (B) Luciferase reporter assays of human embryonic kidney cells 293 co‐transfected with vector, wild‐type 3’‐UTR of PPP2CA (PPP2CA‐3’UTR‐WT) or mutant 3’‐UTR of PPP2CA (PPP2CA‐3’UTR‐MUT), along with mimic negative control (NC) or miR‐409‐3p mimics showed reduced activity only in the group co‐transfected with PPP2CA‐3’UTR‐WT and miR‐409‐3p mimics. Concordantly, in senescent cells, PP2A expression was downregulated at both (C) transcript and (D and E) protein levels, with activation of (F) phosphorylated p38 (p‐p38) and (G) phosphorylated JNK (p‐JNK). n = 4 for each bar. Data are mean ± SEM and analysed using unpaired Student's t test for comparison between groups. *, p < 0.05, ***, p < 0.001 vs. corresponding control. p38, p38 mitogen‐activated protein kinase. JNK, c‐Jun N‐terminal kinase

Article Snippet: The antibodies included phosphorylated p38 mitogen‐activated protein kinase (p‐p38), total p38, phosphorylated c‐Jun N‐terminal kinase (p‐JNK), total JNK, phosphorylated extracellular signal‐regulated kinase (p‐ERK) and total ERK from Cell Signalling Technology.

Techniques: Binding Assay, Luciferase, Transfection, Plasmid Preparation, Mutagenesis, Negative Control, Activity Assay, Expressing, Activation Assay

Journal: Antioxidants

Article Title: Protopanaxadiol-Enriched Rice Extracts Suppressed Oxidative and Melanogenic Activities in Melan-a Cells

doi: 10.3390/antiox12010166

Figure Lengend Snippet: Effects of the transgenic rice seed extracts on the MAPKs (ERK 1/2 and p38) and PI3K/Akt signaling pathways. DJ, #8, and GE concentrations were 100 µg/mL. S-PPD and DMSO concentrations were 700 pg/mL and 0.1%, respectively. Data are shown as means ± standard deviations. Lowercase letters indicate significant differences among treatments at p < 0.05.

Article Snippet: The transferred protein was incubated with antibodies specific to the melanogenesis-related proteins (microphthalmia-associated transcription factor (MITF), tyrosinase-related protein-1 (TRP-1), TRP-2, and tyrosinase) (Santa Cruz Biotechnology, Dallas, TX, USA), mitogen-activated protein kinases (MAPKs: p38 MAPK, phosphorylation (p)-p38 MAPK, extracellular signal-regulated kinase (ERK), and p-ERK) (Cell Signaling, Danvers, MA, USA), and phosphatidylinositol 3-kinase (PI3K)/protein kinase B (Akt and p-Akt; Cell Signaling, Danvers, MA, USA) signaling pathways.

Techniques: Transgenic Assay

Journal: Antioxidants

Article Title: Protopanaxadiol-Enriched Rice Extracts Suppressed Oxidative and Melanogenic Activities in Melan-a Cells

doi: 10.3390/antiox12010166

Figure Lengend Snippet: Schematic diagram of the activation of p-ERK 1/2, p-Akt, and p-p38 on melanogenesis regulation.

Article Snippet: The transferred protein was incubated with antibodies specific to the melanogenesis-related proteins (microphthalmia-associated transcription factor (MITF), tyrosinase-related protein-1 (TRP-1), TRP-2, and tyrosinase) (Santa Cruz Biotechnology, Dallas, TX, USA), mitogen-activated protein kinases (MAPKs: p38 MAPK, phosphorylation (p)-p38 MAPK, extracellular signal-regulated kinase (ERK), and p-ERK) (Cell Signaling, Danvers, MA, USA), and phosphatidylinositol 3-kinase (PI3K)/protein kinase B (Akt and p-Akt; Cell Signaling, Danvers, MA, USA) signaling pathways.

Techniques: Activation Assay

Journal: Drug Design, Development and Therapy

Article Title: Novel targeting of PEGylated liposomes for codelivery of TGF-β1 siRNA and four antitubercular drugs to human macrophages for the treatment of mycobacterial infection: a quantitative proteomic study

doi: 10.2147/DDDT.S79369

Figure Lengend Snippet: Effects of the novel NP-siRNA liposomes on the expression levels of pro- and antiautophagic proteins in THP-1-derived macrophages determined by Western blotting assay. Notes: ( A ) Representive blots of p-PI3K, p-mTOR, mTOR, p-p38 MAPK, p38 MAPK, PTEN, beclin 1, and LC3 in macrophages treated with the NP-siRNA liposomes at 2.5, 12.5, and 62.5 μg/mL for 24 hours. ( B ) Bar graphs show the effect of NP-siRNA liposomes on the levels of p-PI3K, p-mTOR, mTOR, p-p38 MAPK, p38 MAPK, PTEN, beclin 1, and LC3 in macrophages. β-actin was used as the internal control. Data are the mean ± SD of three independent experiments. * P <0.05 by one-way ANOVA followed by Tukey’s post hoc test. Abbreviations: ANOVA, analysis of variance; LC3, microtube-associated protein 1A/1B light chain 3; MAPK, mitogen-activated protein kinase; mTOR, mammalian target of rapamycin; NP, nanoparticle; PI3K, phosphoinositide 3-kinase; PTEN, phosphatase and tensin homologue; SD, standard deviation; siRNA, small interfering RNA.

Article Snippet: Primary antibodies against human phosphorylated phosphoinositide 3-kinase (p-PI3K), phosphorylated p38 mitogenactivated protein kinase (p-p38 MAPK), p38 MAPK, mammalian target of rapamycin (mTOR), p-mTOR, phosphorylated extracellular signal-regulated kinase (ERK), p-ERK, phosphatase and tensin homologue (PTEN), beclin 1, microtubule-associated protein 1A/1B-light chain 3 (LC3), nuclear factor-κB (NF-κB), and nuclear factor (erythroid-derived 2)-like 2 (Nrf2) were purchased from Cell Signaling Technology Inc. (Beverly, MA, USA).

Techniques: Expressing, Derivative Assay, Western Blot, Standard Deviation, Small Interfering RNA

Journal: Molecular Pain

Article Title: Cannabinoid-mediated modulation of neuropathic pain and microglial accumulation in a model of murine type I diabetic peripheral neuropathic pain

doi: 10.1186/1744-8069-6-16

Figure Lengend Snippet: During 8 months of diabetes, Western blotting (A) identified mild increasing levels of Iba1 (B), beginning at 3 months of diabetes, when compared to non-diabetic littermates . As well, phosphorylated p38 MAPK elevates during diabetes until 5 months of time ( C ). Sample protein blots are demonstrated in A from a total of 3 sample blots identified for each marker and each time point. Multiple ANOVA tests were performed in each case, with * indicating significant difference (p < 0.05) between diabetic and non-diabetic cohorts.

Article Snippet: Primary antibodies used were goat anti-ionized calcium-binding adaptor molecule 1 (Iba-1; 1:1000; Abcam, Cambridge, MA) for microglial identification, rabbit anti-cannabinoid receptor 1 antibody (CB1 receptor, 1:500, Sigma Aldrich Canada), rabbit anti-cannabinoid receptor 2 antibody (CB2 receptor, 1:500, Sigma Aldrich Canada), rabbit anti-phosphorylated p38 Mitogen Activated Protein Kinase (p-p38 MAPK; 1:100; Cell Signalling Technology, USA), and rabbit anti-microtubule associated protein-2 antibody produced in rabbit (MAP-2, 1:500, M3696, Sigma Aldrich Canada) for neuronal identification.

Techniques: Western Blot, Marker

Journal: Molecular Pain

Article Title: Cannabinoid-mediated modulation of neuropathic pain and microglial accumulation in a model of murine type I diabetic peripheral neuropathic pain

doi: 10.1186/1744-8069-6-16

Figure Lengend Snippet: During 5 months of diabetes, Western blotting (A) identified mild increased levels of Iba1 (B) in diabetic mice . As well, phosphorylated p38 MAPK was elevated during diabetes at 5 months of time ( C ). Sample protein blots are demonstrated in A (total of 3 sample blots identified for each marker and each intervention at the final time point). Only intranasal or intraperitoneal cannabidiol administered at the onset of diabetes was associated with suppression of Iba1 and phosphorylated p38 MAPK levels at endpoint ( B, C ). Multiple ANOVA tests were performed in each case, with * indicating significant difference (p < 0.05) between diabetic cohorts receiving intranasal cannabidiol and no intervention after 5 months of diabetes.

Article Snippet: Primary antibodies used were goat anti-ionized calcium-binding adaptor molecule 1 (Iba-1; 1:1000; Abcam, Cambridge, MA) for microglial identification, rabbit anti-cannabinoid receptor 1 antibody (CB1 receptor, 1:500, Sigma Aldrich Canada), rabbit anti-cannabinoid receptor 2 antibody (CB2 receptor, 1:500, Sigma Aldrich Canada), rabbit anti-phosphorylated p38 Mitogen Activated Protein Kinase (p-p38 MAPK; 1:100; Cell Signalling Technology, USA), and rabbit anti-microtubule associated protein-2 antibody produced in rabbit (MAP-2, 1:500, M3696, Sigma Aldrich Canada) for neuronal identification.

Techniques: Western Blot, Marker

Journal: Journal of Inflammation (London, England)

Article Title: Signaling pathways involved in LPS induced TNFalpha production in human adipocytes

doi: 10.1186/1476-9255-7-1

Figure Lengend Snippet: Activation of p38 MAP Kinase and NFkappaB pathways regulate LPS induced TNFalpha synthesis from human mature adipocytes . Panel A: Effect of NFkappaB and p38 inhibitors on TNFalpha secretion . The concentrations of TNFalpha (pg/mL) were determined at 6 hours in the medium of mature adipocyte cultures treated or not with: 1 μg/mL of LPS alone; LPS and increasing concentrations of NFkappaB inhibitor (NFkBi 0.1, 0.5 and 1 μM); LPS and increasing concentrations of p38 MAP kinase inhibitor (SB, 0.1, 0.5, 1 and 5 μM); LPS and NFkBi (1 μM) + SB (1 μM). The graph represents the mean ± SE of the results from one patient (n = 6 for each condition), representative of three experiments on three different patients. ***P < 0.001%, *P < 0.05%, versus cells treated with LPS alone. Panel B: TNFalpha gene expression . TNFalpha gene expression was determined at 4, 5 and 6 hours of treatment in mature adipocyte cultures, treated or not with: 1 μg/mL of LPS alone; LPS and NFkappaB inhibitor (NFkBi, 1 μM); LPS and p38 MAP kinase inhibitor (SB, 1 μM); LPS and NFkBi (1 μM) + SB (1 μM). The graph represents the mean ± SD of the results from one patient (n = 5 for each condition), representative of two experiments on two different patients. ***P < 0.001%, *P < 0.05%, versus cells treated with LPS alone.

Article Snippet: Human phosphorylated p38 MAP Kinase protein was detected with anti-phospho-p38 MAP Kinase antibody (Thr180/Tyr182) (Cell Signaling, Ozyme, Saint Quentin Yvelines, France) antibody at a 1/1000 dilution.

Techniques: Activation Assay, Expressing

Journal: Journal of Inflammation (London, England)

Article Title: Signaling pathways involved in LPS induced TNFalpha production in human adipocytes

doi: 10.1186/1476-9255-7-1

Figure Lengend Snippet: p38 MAP Kinase phosphorylation by LPS . Mature adipocyte cells were treated with LPS at 1 μg/mL for 5, 10 and 20 min, or with LPS + p38 MAP Kinase inhibitor (SB, 1 μM) for 5 min. Proteins (50 μg per lane) were separated by SDS-PAGE and analyzed by Western blotting using an anti-phospho-p38 MAP Kinase protein antibody (Thr180/Tyr182, panel B ). Loading equality was controlled using antibody against the unphosphorylated isoform of p38 ( panel A ). The data represent a typical result from two independent experiments.

Article Snippet: Human phosphorylated p38 MAP Kinase protein was detected with anti-phospho-p38 MAP Kinase antibody (Thr180/Tyr182) (Cell Signaling, Ozyme, Saint Quentin Yvelines, France) antibody at a 1/1000 dilution.

Techniques: SDS Page, Western Blot

Journal: Journal of Inflammation (London, England)

Article Title: Signaling pathways involved in LPS induced TNFalpha production in human adipocytes

doi: 10.1186/1476-9255-7-1

Figure Lengend Snippet: Signaling pathways involved in LPS induced TNFalpha production in human adipocytes . LPS, with LBP and CD14 (coming from medium) bind to TLR4 on mature human adipose cells. Binding activates two main pathways in the cells. One pathway leads to the activation of NFkappaB (through TRAF6 and IKK). The other pathway passes through phosphorylated p38 MAP Kinase. Both pathways enable the activation of TNFalpha transcription, followed by cleavage of the protein via a membrane metalloprotease, ADAM17 or TACE, leading to the release of the soluble form of TNFalpha. The PI3K represents a third pathway, which activates NFkappaB and p38 MAPK. Another kinase, maybe the PI4K, plays an inhibitory role in the LPS activation of these 2 pathways. As far as the PKC is concerned, it probably activates IKK, or the p38 MAPKs, or both pathways. However, activation is only visible once the PI4K is inactive. It is therefore possible that PI4K constitutively inhibits PKC.

Article Snippet: Human phosphorylated p38 MAP Kinase protein was detected with anti-phospho-p38 MAP Kinase antibody (Thr180/Tyr182) (Cell Signaling, Ozyme, Saint Quentin Yvelines, France) antibody at a 1/1000 dilution.

Techniques: Binding Assay, Activation Assay

Journal: PLoS ONE

Article Title: CD44 Plays a Critical Role in Regulating Diet-Induced Adipose Inflammation, Hepatic Steatosis, and Insulin Resistance

doi: 10.1371/journal.pone.0058417

Figure Lengend Snippet: The percentages of different CD4/CD8 cell populations were determined by FACS analysis. The percentage of CD8 single-positive T lymphocytes (CD4 − CD8 + ) was decreased in WAT of CD44KO(HFD) compared to WT (HFD) mice. (n = 5–6 mice per each group). Data represent mean±SEM. *p<0.05, ***p<0.001. (B) Protein lysates were extracted from WAT of WT(HFD) and CD44KO(HFD) mice, and Western blot analysis was performed using phosphorylated or total p38 MAPK antibodies and phosphorylated JNK antibody.

Article Snippet: Proteins were subsequently examined by Western blot analysis using antibodies against phosphorylated p38 MAP kinase (p-p38), phosphorylated JNK (p-JNK), and total p38 MAP kinase (p38) (Cell Signaling Technology).

Techniques: Western Blot