Journal: Frontiers in Pharmacology

Article Title: An Integrated Proteomics and Bioinformatics Approach Reveals the Anti-inflammatory Mechanism of Carnosic Acid

doi: 10.3389/fphar.2018.00370

Figure Lengend Snippet: CA restrained the activation of ERK, JNK, and p38 MAPKs in LPS-challenged RAW264.7 cells. Cells were treated with LPS (1 μg/ml) with or without CA (5, 10, and 20 μM) for 1 h. (A) Phosphorylations of ERK, JNK, and p38 protein were determined by western blot assay. (B–D) Quantitative analysis for relative phosphorylation levels of ERK (B) , JNK (C) , and p38 MAPK (D) was performed by normalizing to the control group. Data are expressed as mean ± SEM from three individual experiments. ∗ P < 0.05, ∗∗ P < 0.01 vs. LPS group. ## P < 0.01 vs. control group by ANOVA with Bonferroni’s post hoc test.

Article Snippet: Antibodies against COX2 (12282), GAPDH (3683), p-IKKα/β (2697), IKKα (11930), IKKβ (8943), p-IκB-α (2859), IκB-α (4814), p-NF-κB p65 (3033), NF-κB p65 (8242), p-ERK1/2 MAPK (4370), p-SAPK/JNK (4668), SAPK/JNK (9252), p-p38 MAPK (4511), p38 MAPK (8690), Histone H3 (4499), α-Tubulin (9099), rabbit IgG (7074), and mouse IgG (7076) were purchased from Cell Signaling Technology (Beverly, MA, United States).

Techniques: Activation Assay, Western Blot, Phospho-proteomics, Control

Journal: Ecotoxicology and environmental safety

Article Title: Arsenic trioxide induces expression of BCL-2 expression via NF-κB and p38 MAPK signaling pathways in BEAS-2B cells during apoptosis.

doi: 10.1016/j.ecoenv.2021.112531

Figure Lengend Snippet: Fig. 3. Upregulation of BCL-2 expression by As2O3 through NF-κB activation and the p38 MAPK pathway in BEAS-2B cells. a. BEAS-2B cells were incubated with or without 2.0 µM As2O3 for 6 h, and they were stained with anti-NF-κB antibody and DAPI. Green fluorescence represents NF-κB and blue fluorescence represents nuclear DAPI staining. The bar represents 50 µm. b and c. BEAS-2B cells pretreated with 10 μM BAY11–7082 for 30 min, were incubated with or without 2.0 µM As2O3 for 24 h. Levels of BCL-2 mRNA (b) and protein (c) expression were measured using semi-quantitative RT-PCR analysis and Western blotting analysis, respectively. d. BEAS-2B cells were incubated with 2.0 µM of As2O3 for 24 h. Cell lysates were prepared and subjected to SDS-PAGE followed by immunoblotting using anti-p-ERK, anti-ERK, anti-p-p38 MAPK, anti-p38 MAPK, anti-p-JNK, and anti-JNK antibodies. e and f. BEAS-2B cells pretreated with 20 μM PD98059, 25 μM SP600125 or 10 μM SB203580 for 30 min were incubated with or without 2.0 µM As2O3 for 24 h. The levels of BCL-2 mRNA (e) and protein (f) expressions were measured by semi-quantitative RT-PCR analysis and Western blotting analysis, respectively. The band intensities representing BCL-2 protein expression levels were quantitated using the Image J Gel Analysis program and normalized by the intensity of the β-actin internal control. **p < 0.01, significantly different from the control group; ##p < 0.01, significantly different from the As2O3-treatment group.

Article Snippet: Anti-poly (ADP-ribose) polymerase (PARP; 9542), anti-ERK1/2 (4695), anti-p-ERK1/2 (phosphorylated at Thr202/Tyr204, 4370), anti-JNK (9358), anti-p-JNK (phosphorylated at Thr183/Tyr185, 9255), anti-p38 MAPK (8690), and anti-p-p38 MAPK (phosphorylated at Thr180/Tyr182, 4511) were ordered from Cell Signaling Technology Comp.

Techniques: Expressing, Activation Assay, Incubation, Staining, Fluorescence, Quantitative RT-PCR, Western Blot, SDS Page, Control

Journal: Ecotoxicology and environmental safety

Article Title: Arsenic trioxide induces expression of BCL-2 expression via NF-κB and p38 MAPK signaling pathways in BEAS-2B cells during apoptosis.

doi: 10.1016/j.ecoenv.2021.112531

Figure Lengend Snippet: Fig. 6. A proposed model for As2O3-induced apoptosis of BEAS-2B cells showing the involvement of BCL-2 expres sion and nuclear translocation. As2O3 induces NF-κB transportation into the nucleus. The NF-κB and p38 MAPK signaling pathways upregulate the expression of BCL-2 mRNA and protein. Because of insufficient FKBP38, BCL- 2 in the cytoplasm is shuttled into the nucleus, where it blocks transcription factor activity and initiates apoptosis due to the decreased transcription factor activity.

Article Snippet: Anti-poly (ADP-ribose) polymerase (PARP; 9542), anti-ERK1/2 (4695), anti-p-ERK1/2 (phosphorylated at Thr202/Tyr204, 4370), anti-JNK (9358), anti-p-JNK (phosphorylated at Thr183/Tyr185, 9255), anti-p38 MAPK (8690), and anti-p-p38 MAPK (phosphorylated at Thr180/Tyr182, 4511) were ordered from Cell Signaling Technology Comp.

Techniques: Translocation Assay, Protein-Protein interactions, Expressing, Activity Assay

Journal: Molecular Medicine Reports

Article Title: O-GlcNAcylation contributes to intermittent hypoxia-associated vascular dysfunction via modulation of MAPKs but not CaMKII pathways

doi: 10.3892/mmr.2021.12384

Figure Lengend Snippet: CIH enhances O-GlcNAc levels, and the expression levels of OGT, p-p38 MAPK and p-ERK1/2 levels, but decreases OGA expression in mesenteric arteries. The groups were as follows: i) CON, normoxic (21% O 2 ) condition; and ii) CIH, intermittent hypoxia cycles (6–8% O 2 for 2 min and 21% O 2 for 2 min). Data are presented as the mean ± SD (n=3-5). *P<0.05 vs. CON group. CIH, chronic intermittent hypoxia; O-GlcNAc, O-linked-β-N-acetylglucosamine; OGT, O-GlcNAc transferase; OGA, O-GlcNAcase; p-, phosphorylated; CON, control; CaMKII, Ca 2+ /calmodulin-dependent kinase II; t-, total protein.

Article Snippet: PugNAc (OGA inhibitor; cat. no. sc-204415), primary antibodies for O-GlcNAc (mouse monoclonal; cat. no. sc-59623; 1:800), CaMKII (rabbit polyclonal; cat. no. sc-9035; 1:800) and phosphorylated (p)-CaMKII (mouse monoclonal; cat. no. sc-32289; 1:800), as well as secondary anti-mouse (cat. no. sc-358914; 1:4,000) and anti-rabbit IgG antibodies (cat. no. sc-2004; 1:4,000) were purchased from Santa Cruz Biotechnology, Inc. Antibodies against OGT (rabbit polyclonal; cat. no. 11576-2-AP; 1:1,000) and OGA (rabbit polyclonal; cat. no. 14711-1-AP; 1:400) were obtained from ProteinTech Group, Inc. Antibodies against p38 MAPK (rabbit polyclonal; cat. no. 9212; 1:1,000), p-p38 MAPK (rabbit monoclonal; cat. no. 4511; 1:1,000), ERK1/2 (rabbit monoclonal; cat. no. 4695; 1:1,000), p-ERK1/2 (rabbit monoclonal; cat no. 4376; 1:1,000) and GAPDH (rabbit monoclonal; cat. no. 5174; 1:3,000) were purchased from Cell Signaling Technology, Inc. SNP solution was freshly prepared.

Techniques: Expressing

Journal: Molecular Medicine Reports

Article Title: O-GlcNAcylation contributes to intermittent hypoxia-associated vascular dysfunction via modulation of MAPKs but not CaMKII pathways

doi: 10.3892/mmr.2021.12384

Figure Lengend Snippet: Protein O-GlcNAc levels interfere with the phosphorylation of p38 MAPK, ERK1/2 and CaMKII. (A) Protein levels of O-GlcNAc, OGA, OGT, p-p38 MAPK, p-ERK1/2 and p-CaMKII in cultured aortas that received either AON or AIH treatment in the presence of DMSO, PugNAc or ST045849. (B) Protein levels of O-GlcNAc, OGA, OGT, p-p38 MAPK, p-ERK1/2 and p-CaMKII in cultured aortas from the CON or CIH rats in the presence of DMSO, PugNAc or ST045849. Experimental groups were as follows: i) AON, 3-h normoxia treatment; ii) AIH, 3-h intermittent hypxia treatment; iii) CON, normoxic (21% O 2 ) condition; and iv) CIH, intermittent hypoxia cycles (6–8% O 2 for 2 min and 21% O 2 for 2 min). Data are presented as the mean ± SD (n=3). *P<0.05. O-GlcNAc, O-linked-β-N-acetylglucosamine; CaMKII, Ca 2+ /calmodulin-dependent kinase II; OGA, O-GlcNAcase; OGT, O-GlcNAc transferase; AIH, acute intermittent hypoxia; CON, control; CIH, chronic intermittent hypoxia; p-, phosphorylated; t-, total protein.

Article Snippet: PugNAc (OGA inhibitor; cat. no. sc-204415), primary antibodies for O-GlcNAc (mouse monoclonal; cat. no. sc-59623; 1:800), CaMKII (rabbit polyclonal; cat. no. sc-9035; 1:800) and phosphorylated (p)-CaMKII (mouse monoclonal; cat. no. sc-32289; 1:800), as well as secondary anti-mouse (cat. no. sc-358914; 1:4,000) and anti-rabbit IgG antibodies (cat. no. sc-2004; 1:4,000) were purchased from Santa Cruz Biotechnology, Inc. Antibodies against OGT (rabbit polyclonal; cat. no. 11576-2-AP; 1:1,000) and OGA (rabbit polyclonal; cat. no. 14711-1-AP; 1:400) were obtained from ProteinTech Group, Inc. Antibodies against p38 MAPK (rabbit polyclonal; cat. no. 9212; 1:1,000), p-p38 MAPK (rabbit monoclonal; cat. no. 4511; 1:1,000), ERK1/2 (rabbit monoclonal; cat. no. 4695; 1:1,000), p-ERK1/2 (rabbit monoclonal; cat no. 4376; 1:1,000) and GAPDH (rabbit monoclonal; cat. no. 5174; 1:3,000) were purchased from Cell Signaling Technology, Inc. SNP solution was freshly prepared.

Techniques: Cell Culture

Journal: Nature Communications

Article Title: JNK regulates muscle remodeling via myostatin/SMAD inhibition

doi: 10.1038/s41467-018-05439-3

Figure Lengend Snippet: SMAD-linker and JNK phosphorylation with exercise and muscle contraction in mice. a ICR mice underwent moderate intensity treadmill running for 15, 30, or 60 min and gastrocnemius muscles were collected. Control (rest) mice did not undergo treadmill running. Western blotting was used to determine exercise-induced signal transduction. Data from N = 3 mice/group are shown. b Electrodes were used to stimulate the lower hindlimb muscles from ICR mice [C; contracted]. The contralateral limb was unstimulated and acted as a control [B; basal]. Data from N = 3 mice/group are shown. c Both soleus muscles were rapidly removed from mice and attached to a force tranducer in oxygenated Kreb’s Henseleit Buffer. One muscle from each mouse was left at resting tension and acted as a basal control [Basal; B], while contralateral muscle was stretched for 10 min at a force of 0.12 N [Stretched; S]. N = 6 independent experiments were performed, and individual results from N = 3 are shown. pSMAD2L, linker region phosphorylated SMAD2; pJNK, phosphorylated (active) C-Jun N-terminal Kinase; pAMPK, phosphorylated (T172) AMP-activated protein kinase; pERK, phosphorylated extracellular signal regulated kinase; pP38, phosphorylated P38 Mitogen-Activated Protein Kinase; SMAD2, Total SMAD2. Images obtained using stain-free gel technology (Bio-Rad) that allows for total protein visualization and quantification are shown as a loading control (Loading)

Article Snippet: The following antibodies were used for the detection of phosphorylated and total protein levels: pSMAD2-C (CST 8828), pSMAD2-L (CST 3104), pSMAD3-L (PA5-38521), pERK (CST 4370), SMAD 2 (CST 5339), SMAD 2/3 (CST 8685), pJNK (Promega V7931 or CST4668), JNK Total (CST 9252), α-Tubulin (CST 3873), GAPDH (CST5174), pAkt (CST 9271), pP38 (CST 4511), pAMPK (CST 2531).

Techniques: Phospho-proteomics, Muscles, Control, Western Blot, Transduction, Staining

Journal: Nature Communications

Article Title: JNK regulates muscle remodeling via myostatin/SMAD inhibition

doi: 10.1038/s41467-018-05439-3

Figure Lengend Snippet: JNK is the upstream kinase for SMAD2 linker phosphorylation in muscle. a – c EDL and TA muscles from wild-type control (WT; MCK-Cre −/+ ) and muscle-specific JNK1/2 knockout mice (KO) were stimulated via in vitro (EDL) and in situ (TA) contraction. Immunoblotting of phosphorylated and total SMAD2 and JNK was performed ( a ). SMAD2-linker (SMAD2L) phosphorylation in response to in vitro contraction ( b ) and in situ contraction ( c ) was blunted in muscle JNK knockout mice. Each data point represents the basal and contraction results from an individual animal joined by a line. * P < 0.05, *** P < 0.01 vs Basal from the same genotype by repeated measures two-way ANOVA and Sidak’s post hoc testing. Main effects of genotype are displayed with P -values. d C2C12 myoblasts were treated with the JNK activator anisomycin (5 μM) for 30 min. e C2C12 myoblasts were transfected with plasmids expressing constitutively active JNK1, JNK2, or a combination of JNK1 and 2. Empty vector (EV) transfected cells were used as a control. f The ability of JNK to directly phosphorylate SMAD2 was assessed using an in vitro kinase assay. C2C12 lysates expressing empty vector (EV), or constitutively active JNK1 or JNK2 were purified by FLAG immunoprecipitation and incubated with recombinant SMAD2 and SMAD3 proteins. For tissue culture experiments ( d – f ), three independent experiments were performed and the data from one representative experiment, including all replicates is displayed. In all experiments, JNK activation (pJNK) and SMAD2 linker phosphorylation (pSMAD2L) were assessed by immunoblotting. Images obtained using stain-free gel technology (Bio-Rad) that allows for total protein visualization and quantification are shown as a loading control (Loading). pJNK phosphorylated (active) C-Jun N-terminal Kinase, pSMAD2L, linker-region phosphorylated SMAD2; SMAD2, total SMAD2; pP38, phosphorylation P38 MAPK; pERK, phosphorylated extracellular signal regulated kinase

Article Snippet: The following antibodies were used for the detection of phosphorylated and total protein levels: pSMAD2-C (CST 8828), pSMAD2-L (CST 3104), pSMAD3-L (PA5-38521), pERK (CST 4370), SMAD 2 (CST 5339), SMAD 2/3 (CST 8685), pJNK (Promega V7931 or CST4668), JNK Total (CST 9252), α-Tubulin (CST 3873), GAPDH (CST5174), pAkt (CST 9271), pP38 (CST 4511), pAMPK (CST 2531).

Techniques: Phospho-proteomics, Muscles, Control, Knock-Out, In Vitro, In Situ, Western Blot, Transfection, Expressing, Plasmid Preparation, Kinase Assay, Purification, Immunoprecipitation, Incubation, Recombinant, Activation Assay, Staining