Journal: Journal of neurochemistry

Article Title: Altered MAP kinase phosphorylation and impaired motor coordination in PTPRR deficient mice.

doi: 10.1111/j.1471-4159.2006.04398.x

Figure Lengend Snippet: Fig. 3 Altered MAPK phosphorylation levels in Ptprr)/) mouse brain. Degree of ERK1/2 (a), p38 (b) and JNK1/2 (c) biphosphorylation in cerebellum and cerebrum of wildtype (+/+, white bars) and Ptprr)/) ()/ ), black bars) mice as determined by Western analyses of tissue lysates from individual mice (n = 6 for each tissue and genotype group) using phospho-specific antibodies. Representative examples (three out of the six for each tissue and genotype group) of phos- phorylated ERK1/p44 and ERK2/p42 (pERK) and total ERK1/2 (tot- ERK) immunofluorescent signals are depicted above the corresponding bars. Phosphorylated ERK1/2, p38 and JNK1/2 signals were normalized to total ERK1/2, p38 and JNK1/2 levels, respectively, and plotted as relative phosporylation levels. Results are the mean ± SEM of six samples per group and are representative of at least three independent experiments (*p < 0.0001, **p < 0.01).

Article Snippet: Polyclonal antisera against total ERK1 (1 : 8000), p38 (1 : 2000), and JNK1 (1 : 500) were obtained from Santa Cruz Biotechnology (sc-93, sc-535 and sc-474, respectively; Santa Cruz, CA, USA), antiserum against total MEK1 (1 : 1000) was from Biosource (AH01102), monoclonal antibody ERK-PT115 (1 : 500) against threonine-phosphorylated ERK1/2 was from Sigma-Aldrich (St Louis, MO, USA) (M7802), antiserum against Elk-1 (1 : 1000) and monoclonal phospho-specific antibodies against ERK1/2 (1 : 2000), p38 (1 : 2000), JNK1 (1 : 500), MEK1/2 (1 : 1000), Elk-1 (1 : 1000) and S6 ribosomal protein (1 : 500) were from Cell Signalling Technology (Beverly, MA, USA) (nos 9182, 9106, 9216, 9255, 9121, 9186 and 4856, respectively).

Techniques: Phospho-proteomics, Western Blot

Journal: Antioxidants

Article Title: Differences in Airway Remodeling and Emphysematous Lesions between Rats Exposed to Smoke from New-Type and Conventional Tobacco Varieties

doi: 10.3390/antiox13050511

Figure Lengend Snippet: Cigarette smoke-altered Nrf2 and p38 MAPK expression in the lung tissues of rats. Immunohistochemical staining of Nrf2 ( A – D ), p38 MAPK ( E – H ) and phospho-p38MAPK ( I – L ). Representative images at 60 days. 200×, scale bars = 50 µm. The relative expression levels of Nrf2 ( M ), p38 MAPK ( N ) and phosphor-p38 MAPK ( O ). The ratio of phosphor-p38 to p38 ( P ). Results are presented as means with standard deviations, as shown by the vertical bars (n = 6). The statistical significance between all data is compared. Bars with the same letter(s) indicate no significant differences at the level of p < 0.05.

Article Snippet: Rabbit polyclonal antibodies against Bcl-2 (26593-1-AP), Nrf2 (16396-1-AP), p38 MAPK (14064-1-AP) and phospho-p38 MAPK (Thr180/Tyr182) (28796-1-AP) were purchased from Proteintech (Wuhan, China).

Techniques: Expressing, Immunohistochemical staining, Staining

Journal: Biochemical Journal

Article Title: Peroxiredoxin 1 (Prx1) is a dual-function enzyme by possessing Cys-independent catalase-like activity

doi: 10.1042/BCJ20160851

Figure Lengend Snippet: ( a–e ) Western blot analysis was conducted on total protein extracts from cells transfected with various TnPrx1 constructs and treated with various concentrations of exogenous H 2 O 2 using antibodies specific to p38 and phosphorylated p38 (p-p38), respectively. Blank control cells received no transfection. Representative data from one of the three or more independent experiments were shown. Note: Antibody to p38 recognized a single band at 43 kDa, while antibody to p-p38 also produced a nonspecific band (NS) above the major band at 43 kDa for each sample. The NS bands were much weaker than—but proportional to—the major bands, a finding that was also observed by other investigators (e.g. [ – ]). Bar charts showed relative density (RD) of p-p38 major bands (vs. p38). The error bars represented standard deviations (SDs) from three replicated blots. An asterisk in each bar chart indicates the lowest concentration of exogenous H 2 O 2 starting to give statistical significance between H 2 O 2 -treated and untreated controls (* P < 0.05 by Student's t -test); ( f ) Confirmation on the protein expression from TnPrx1 constructs by Western blot analysis using antibodies specific to His-tag and to human GAPDH control, respectively. RD = relative density; POX = Cys-dependent thioredoxin peroxidase; CAT = Catalase-like activity of peroxiredoxin 1.

Article Snippet: This was followed by Western blot analysis using antibodies against p38 (1:1000 dilution; Catalog No. 8690, Cell Signaling Technology, Danvers) and phosphorylated p38 protein (p-p38) (1:1000 dilution; Catalog No. 4511, Cell Signaling Technology, Danvers), respectively.

Techniques: Western Blot, Transfection, Construct, Control, Produced, Concentration Assay, Expressing, Activity Assay

Journal: Biochemical Journal

Article Title: Peroxiredoxin 1 (Prx1) is a dual-function enzyme by possessing Cys-independent catalase-like activity

doi: 10.1042/BCJ20160851

Figure Lengend Snippet: ( a–e ) Western blot analysis of total protein extracts from cells co-transfected with HsPrx1 -siRNA-402 and various TnPrx1 constructs after the treatment of various concentrations of exogenous H 2 O 2 using antibodies specific to p38 and phosphorylated p38 (p-p38) proteins. Control cells were transfected with siRNA-402 only. Representative data from one of the three independent experiments are shown. ( f ) Protein expression of TnPrx1 constructs by Western blot analysis using antibodies specific to His-tag and to human GAPDH (glyceraldehyde-3-phosphate dehydrogenase) (control). An asterisk in each bar chart indicates the lowest concentration of exogenous H 2 O 2 yielding statistical significance between H 2 O 2 -treated cells and untreated controls (* P < 0.05, ** P < 0.01 by Student's t -test). RD = relative density.

Article Snippet: This was followed by Western blot analysis using antibodies against p38 (1:1000 dilution; Catalog No. 8690, Cell Signaling Technology, Danvers) and phosphorylated p38 protein (p-p38) (1:1000 dilution; Catalog No. 4511, Cell Signaling Technology, Danvers), respectively.

Techniques: Western Blot, Transfection, Construct, Control, Expressing, Concentration Assay

Journal: Journal of pharmacological sciences

Article Title: Nafamostat protects against early brain injury after subarachnoid hemorrhage in mice.

doi: 10.1016/j.jphs.2021.10.007

Figure Lengend Snippet: Fig. 4. The effect of nafamostat on human brain microvascular endothelial cells (HBMVECs) after thrombin and hypoxia stress. (A) The experimental protocol used to assess the effects of nafamostat against combined thrombin and hypoxia stress in HBMVECs. The effect of nafamostat on phosphorylation of p38 (B) and expression of ICAM-1 (C) 24 h after the thrombin and hypoxia stress. Data are expressed as means ± standard errors of the mean. n ¼ 5 or 6 per group. ##p < 0.01, sham vs. vehicle, Student's t-test. *p < 0.05, **p < 0.01, vs. vehicle, Dunnett's test.

Article Snippet: The following primary antibodies were used: rabbit anti-thrombin (1:1000; Abcam;MA, USA), rabbit anti-prothrombin (1:1000; Abcam), rabbit anti-matrix metalloproteinase (MMP-9) (1:1000; Abcam), rabbit anti-phospho-p38 (1:1,000, Cell Signaling Technology, MA, USA), rabbit anti p38 (1:1,000, Cell Signaling Technology), rabbit antiintercellular adhesion molecule (ICAM-1) (1:1,000, Cell Signaling Technology), and monoclonal anti-b-actin (1:2000; SigmaeAldrich).

Techniques: Phospho-proteomics, Expressing

Journal: Biochemical and biophysical research communications

Article Title: AMPK activators contribute to maintain naïve pluripotency in mouse embryonic stem cells.

doi: 10.1016/j.bbrc.2018.11.164

Figure Lengend Snippet: Fig. 4. p38 is a functional downstream of AMPK. (A) p38 activation with AMPK activators. Western blot analysis shows protein expression of p38, phosphorylated p38 (Thr180/ Tyr182), a unique p38 downstream target mapkapk2 and phosphorylated mapkapk2 (Thr334). Note that p38 is activated commonly with AMPK activators. (B and C) FACS analysis for Rex1-GFPþcell appearance with a p38 inhibitor. p38i: SB203580 (10 mM). (D) Tetracycline-inducible (Tet-ON) constitutive active form of p38 (CA-p38) expression marked with mCherry. Western blot confirms dox-inducible activation of p38 pathway with CA-p38 expression. Doxycycline (1 mg/ml) treatment. (E) FACS analysis for Rex1-GFPþcell appearance after p38 activation.

Article Snippet: Please cite this article as: Y. Liu, J.K. Yamashita, AMPK activators contrib Biochemical and Biophysical Research Communications, https://doi.org/1 primary antibodies for following targets: phosphorylated-AMPK (Thr172, Cell Signaling (2531S) 1:1000), AMPK (Cell Signaling (2532S), 1:1000), phosphorylated-p38 (Thr180/Tyr182, Cell Signaling (9215S), 1:1000), p38 (Cell Signaling (9212S), 1:1000), phosphorylated-Mapkapk2 (Thr334, Cell Signaling (3007S), 1:1000), Mapkapk2 (Cell Signaling (3042S), 1:1000), b-actin (Sigma (A5441), 1:10000).

Techniques: Functional Assay, Activation Assay, Western Blot, Expressing

Journal: iScience

Article Title: Metabolic orchestration of NOD1 signaling by AMPK-mediated phosphorylation of ZDHHC5

doi: 10.1016/j.isci.2026.115245

Figure Lengend Snippet: Cellular energy stress suppresses PGN-induced NOD1 signaling (A) Mouse BMDM cells were treated with or without glucose for 6 h, then stimulated with C12-iE-DAP (1 μg/mL) for 30 min, p65 and p38 phosphorylation were analyzed by immunoblotting. (B) Mouse BMDM cells were treated with 2-DG (25 mM) in glucose-free medium for 6 h, then stimulated with C12-iE-DAP (1 μg/mL) for 30 min, p65 and p38 phosphorylation were analyzed by immunoblotting. (C) Mouse BMDM cells were treated with metformin (2 mM) for 6 h, then stimulated with C12-iE-DAP (1 μg/mL) for 30 min, p65 and p38 phosphorylation were analyzed by immunoblotting. (D) Mouse iBMDM cells were treated with or without glucose and stimulated with C12-iE-DAP (5 μg/mL) for 7 h. The IL-6 release in the medium was measured with ELISA. For each experimental group, three supernatant samples were analyzed. ∗∗ p < 0.01, NS, p > 0.05. mean ± s.d., Student’s t test. (E) Mouse iBMDM cells were treated with 2-DG (25 mM) in glucose-free medium and stimulated with C12-iE-DAP (5 μg/mL) for 7 h. The IL-6 release in the medium was measured with ELISA. For each experimental group, three supernatant samples were analyzed. ∗∗ p < 0.01, NS, p > 0.05. mean ± s.d., Student’s t test. (F) Mouse iBMDM cells were pre-treated with DMSO or Compound C (5 μM) and treated with or without glucose for 6 h, then stimulated with C12-iE-DAP (1 μg/mL) for 30 min, p65 and p38 kinase phosphorylation were analyzed by immunoblotting. (G) Mouse iBMDM cells were pre-treated with DMSO or Compound C (5 μM) and then treated with or without glucose and stimulated with C12-iE-DAP (5 μg/mL) for 7 h. The IL-6 release in the medium was measured with ELISA. For each experimental group, three supernatant samples were analyzed. ∗∗ p < 0.01, NS, p > 0.05. mean ± s.d., Student’s t test. (H) Mouse iBMDM cells were treated with MK-8722 (2 μM) for 8 h, then stimulated with C12-iE-DAP (1 μg/mL) for 30 min, p65 and p38 phosphorylation were analyzed by immunoblotting. (I) HEK293T cells were treated with or without glucose for 6 h. Representative fluorescence images show the localization of GFP-NOD1 were presented. Scale bar = 10 μm for all images. (J) HEK-293T cells expressing FLAG-NOD1 were treated with or without glucose for 6h. Total, cytosolic, and membrane fractions were immunoblotted with the indicated antibodies. (K) HEK293T cells were pre-treated with DMSO or Compound C (5 μM) and then treated with glucose starvation for 6h. Representative fluorescence images show the localization of GFP-NOD1 were presented. Scale bar = 10 μm for all images. (L) HEK-293T cells expressing FLAG-NOD1 were pre-treated with DMSO or Compound C (5 μM) and treated with or without glucose for 6h. Total, cytosolic, and membrane fractions were immunoblotted with the indicated antibodies.

Article Snippet: Rabbit-Anti-Phospho-p38 MAPK (Thr180/Tyr182) , Cell Signaling Technology , Cat# 4511; RRID: AB_2139682.

Techniques: Phospho-proteomics, Western Blot, Enzyme-linked Immunosorbent Assay, Fluorescence, Expressing, Membrane

Journal: iScience

Article Title: Metabolic orchestration of NOD1 signaling by AMPK-mediated phosphorylation of ZDHHC5

doi: 10.1016/j.isci.2026.115245

Figure Lengend Snippet: AMPK-mediated ZDHHC5 phosphorylation inhibits NOD1 activation (A) ZDHHC5-knockout HEK293T cells reconstituted with ZDHHC5 wild-type (WT) or 2A mutant were transfected to express FLAG-NOD1, then treated with metformin (5 mM) and labeled with alk-C16 for 6 h. NOD1 palmitoylation was detected by click chemistry reaction. (B) ZDHHC5-knockout HEK293T cells reconstituted with ZDHHC5 wild-type (WT) or 2A mutant were treated with metformin (5 mM) for 6 h. Representative fluorescence images show the localization of GFP-NOD1 were presented. Scale bar = 10 μm for all images. (C) ZDHHC5-knockout HEK293T cells reconstituted with ZDHHC5 wild-type (WT) or 2A mutant were treated with or without glucose for 6 h. Representative fluorescence images show the localization of GFP-NOD1 were presented. Scale bar = 10 μm for all images. (D) ZDHHC5-knockout HEK293T cells reconstituted with ZDHHC5 wild-type (WT) or 2A mutant were transfected to express FLAG-NOD1, and treated with metformin (5 mM) for 6 h. Total, cytosolic, and membrane fractions were immunoblotted with the indicated antibodies. (E) BMDMs were generated from Zdhhc5 −/− mice, and were reconstituted with ZDHHC5 wild-type (WT) or 2A mutant using lentiviral transduction. The reconstituted BMDM cells were treated with metformin (2 mM) for 6 h, followed by stimulation with C12-iE-DAP (1 μg/mL) for 30 min. p65 and p38 kinase phosphorylation were analyzed by immunoblotting. (F) ZDHHC5-knockdown iBMDMs were reconstituted with ZDHHC5 wild-type (WT) or 2A mutant using lentiviral transduction. The reconstituted iBMDM cells were treated with metformin (2 mM) for 6 h, followed by stimulation with C12-iE-DAP (1 μg/mL) for 30 min, p65 and p38 kinase phosphorylation were analyzed by immunoblotting. (G) ZDHHC5-knockdown iBMDMs were reconstituted with ZDHHC5 wild-type (WT) or 2A mutant using lentiviral transduction. The reconstituted iBMDMs cells were treated with or without glucose and stimulated with C12-iE-DAP (5 μg/mL) for 7 h. The IL-6 release in the medium was measured with ELISA. For each experimental group, three supernatant samples were analyzed. ∗∗ p < 0.01, NS, p > 0.05. mean ± s.d., Student’s t test.

Article Snippet: Rabbit-Anti-Phospho-p38 MAPK (Thr180/Tyr182) , Cell Signaling Technology , Cat# 4511; RRID: AB_2139682.

Techniques: Phospho-proteomics, Activation Assay, Knock-Out, Mutagenesis, Transfection, Labeling, Fluorescence, Membrane, Generated, Transduction, Western Blot, Knockdown, Enzyme-linked Immunosorbent Assay