Structured Review

Cell Signaling Technology Inc p38
Tat-mediated induction of ICAM-1 expression involves MAPK signaling pathways. ( A ) Western blot analysis demonstrated time-dependent activation of ERK, JNK and <t>p38</t> by Tat in HUVECs. ( B ) Inhibition of the ERK, JNK and p38 MAPK pathways by MEK1/2 (U0126, 20 µM), JNK (SP600125, 20 µM) and p38 (SB203580, 20 µM) inhibitors resulted in amelioration of Tat-mediated induction of ICAM-1 expression. ( C ) Pharmacological inhibition of MAPK pathways by MEK1/2 (U0126, 20 µM), JNK (SP600125, 20 µM) and p38 (SB203580, 20 µM) resulted in amelioration of Tat-mediated induction of monocyte adhesion. All the data are presented as mean ± SD of three independent experiments. **p
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Images

1) Product Images from "HIV Tat Induces Expression of ICAM-1 in HUVECs: Implications for miR-221/-222 in HIV-Associated Cardiomyopathy"

Article Title: HIV Tat Induces Expression of ICAM-1 in HUVECs: Implications for miR-221/-222 in HIV-Associated Cardiomyopathy

Journal: PLoS ONE

doi: 10.1371/journal.pone.0060170

Tat-mediated induction of ICAM-1 expression involves MAPK signaling pathways. ( A ) Western blot analysis demonstrated time-dependent activation of ERK, JNK and p38 by Tat in HUVECs. ( B ) Inhibition of the ERK, JNK and p38 MAPK pathways by MEK1/2 (U0126, 20 µM), JNK (SP600125, 20 µM) and p38 (SB203580, 20 µM) inhibitors resulted in amelioration of Tat-mediated induction of ICAM-1 expression. ( C ) Pharmacological inhibition of MAPK pathways by MEK1/2 (U0126, 20 µM), JNK (SP600125, 20 µM) and p38 (SB203580, 20 µM) resulted in amelioration of Tat-mediated induction of monocyte adhesion. All the data are presented as mean ± SD of three independent experiments. **p
Figure Legend Snippet: Tat-mediated induction of ICAM-1 expression involves MAPK signaling pathways. ( A ) Western blot analysis demonstrated time-dependent activation of ERK, JNK and p38 by Tat in HUVECs. ( B ) Inhibition of the ERK, JNK and p38 MAPK pathways by MEK1/2 (U0126, 20 µM), JNK (SP600125, 20 µM) and p38 (SB203580, 20 µM) inhibitors resulted in amelioration of Tat-mediated induction of ICAM-1 expression. ( C ) Pharmacological inhibition of MAPK pathways by MEK1/2 (U0126, 20 µM), JNK (SP600125, 20 µM) and p38 (SB203580, 20 µM) resulted in amelioration of Tat-mediated induction of monocyte adhesion. All the data are presented as mean ± SD of three independent experiments. **p

Techniques Used: Expressing, Western Blot, Activation Assay, Inhibition

2) Product Images from "Neuroprotective effects of Scrophularia buergeriana extract against glutamate-induced toxicity in SH-SY5Y cells"

Article Title: Neuroprotective effects of Scrophularia buergeriana extract against glutamate-induced toxicity in SH-SY5Y cells

Journal: International Journal of Molecular Medicine

doi: 10.3892/ijmm.2019.4139

Effects of Scrophularia buergeriana extract (SBE) on apoptotic protein expression levels in SH-SY5Y cells. (A) The expression of phosphorylated (p)-p38, B-cell lymphoma-2 (Bcl-2), Bcl-2-associated X protein (Bax), cleaved caspase-3 and cleaved poly (adenosine diphosphate (ADP)-ribose) polymerase (PARP) was measured by western blot analysis. (B) The density of the protein bands was quantified and calculated using ImageJ software. p-p38 protein expression levels were normalized to those of p38, and other protein expression levels were normalized to those of β-actin. The data are expressed as the means ± SEM of independent experiments (n=3). * P
Figure Legend Snippet: Effects of Scrophularia buergeriana extract (SBE) on apoptotic protein expression levels in SH-SY5Y cells. (A) The expression of phosphorylated (p)-p38, B-cell lymphoma-2 (Bcl-2), Bcl-2-associated X protein (Bax), cleaved caspase-3 and cleaved poly (adenosine diphosphate (ADP)-ribose) polymerase (PARP) was measured by western blot analysis. (B) The density of the protein bands was quantified and calculated using ImageJ software. p-p38 protein expression levels were normalized to those of p38, and other protein expression levels were normalized to those of β-actin. The data are expressed as the means ± SEM of independent experiments (n=3). * P

Techniques Used: Expressing, Western Blot, Software

Schematic view of Scrophularia buergeriana extract (SBE) regulating reactive oxygen species (ROS) levels and the apoptotic signaling cascade. Excessive glutamate caused ROS generation and induces neuronal cell apoptosis. The generated ROS triggers p38 and Bcl-2-associated X protein (Bax) protein and damage to mitochondrial function. The activated caspase-3 and poly (adenosine diphosphate (ADP)-ribose) polymerase (PARP) proteins result in apoptosis. SBE treatment reduces the production of ROS by regulating the expression of enzymes, including superoxide dismutase (SOD)1, SOD2 and glutathione peroxidase-1 (GPx-1) and also inhibits the apoptotic pathway.
Figure Legend Snippet: Schematic view of Scrophularia buergeriana extract (SBE) regulating reactive oxygen species (ROS) levels and the apoptotic signaling cascade. Excessive glutamate caused ROS generation and induces neuronal cell apoptosis. The generated ROS triggers p38 and Bcl-2-associated X protein (Bax) protein and damage to mitochondrial function. The activated caspase-3 and poly (adenosine diphosphate (ADP)-ribose) polymerase (PARP) proteins result in apoptosis. SBE treatment reduces the production of ROS by regulating the expression of enzymes, including superoxide dismutase (SOD)1, SOD2 and glutathione peroxidase-1 (GPx-1) and also inhibits the apoptotic pathway.

Techniques Used: Generated, Expressing

3) Product Images from "Anti-influenza A virus activity of rhein through regulating oxidative stress, TLR4, Akt, MAPK, and NF-κB signal pathways"

Article Title: Anti-influenza A virus activity of rhein through regulating oxidative stress, TLR4, Akt, MAPK, and NF-κB signal pathways

Journal: PLoS ONE

doi: 10.1371/journal.pone.0191793

Schematic diagram of rhein on inhibition of IAV infection and IAV-mediated ALI. Rhein can inhibit IAV adsorption and replication. Rhein suppresses IAV replication by inhibiting IAV-mediated oxidative stress and activations of TLR4, Akt, p38, JNK MAPK, and NF-κB signal pathways. Meanwhile, inhibition of these signal pathways further reduces the production of inflammatory cytokines and MMPs and finally decreases IAV-induced ALI.
Figure Legend Snippet: Schematic diagram of rhein on inhibition of IAV infection and IAV-mediated ALI. Rhein can inhibit IAV adsorption and replication. Rhein suppresses IAV replication by inhibiting IAV-mediated oxidative stress and activations of TLR4, Akt, p38, JNK MAPK, and NF-κB signal pathways. Meanwhile, inhibition of these signal pathways further reduces the production of inflammatory cytokines and MMPs and finally decreases IAV-induced ALI.

Techniques Used: Inhibition, Infection, Adsorption

Counteracting effects of different agonists on the antiviral activity of rhein. A549 cells were infected with IAV (MOI = 0.001) and treated with or without ribavirin (Rib, 25 μg/mL), rhein (10 μg/mL), TLR2 agonist LAM-MS (10 μg/ml), TLR3 agonist Poly(I:C) (10 μg/ml), TLR4 agonist LPS-B5 (1 μg/ml), oxidant H 2 O 2 (100 μM), Akt agonist IGF-1 (IGF-1 100 ng/ml), ERK agonist EGF (100 ng/ml), p38/JNK agonist anisomycin (10 μM), and NF-κB agonist PMA (1 μg/ml). Ater 48 h p.i., the antiviral activity was determined by a SRB method (A), IAV replication was determined by a qRT-PCR assay (B). The experiment was repeated five times and each experimental condition was performed in triplicate (n = 5). All data shown were mean ± SD. # P
Figure Legend Snippet: Counteracting effects of different agonists on the antiviral activity of rhein. A549 cells were infected with IAV (MOI = 0.001) and treated with or without ribavirin (Rib, 25 μg/mL), rhein (10 μg/mL), TLR2 agonist LAM-MS (10 μg/ml), TLR3 agonist Poly(I:C) (10 μg/ml), TLR4 agonist LPS-B5 (1 μg/ml), oxidant H 2 O 2 (100 μM), Akt agonist IGF-1 (IGF-1 100 ng/ml), ERK agonist EGF (100 ng/ml), p38/JNK agonist anisomycin (10 μM), and NF-κB agonist PMA (1 μg/ml). Ater 48 h p.i., the antiviral activity was determined by a SRB method (A), IAV replication was determined by a qRT-PCR assay (B). The experiment was repeated five times and each experimental condition was performed in triplicate (n = 5). All data shown were mean ± SD. # P

Techniques Used: Activity Assay, Infection, Laser Capture Microdissection, Mass Spectrometry, Sulforhodamine B Assay, Quantitative RT-PCR

Effects of rhein on TLRs, Akt, MAPK and NF-κB signaling pathways after IAV infection. The treatment of the blank control (BC), negative control (NC), positive control (PC), and rhein-treated group (rhein) was same with that of the “antioxidant assay”. After 48 h p.i., the cells were harvested. The expressions of TLR2, TLR3, and TLR4, the phosphorylations of Akt, ERK, p38, JNK MAPK, and the nuclear translocation of NF-κB p65 were determined by western blotting assay. The experiment was repeated five times (n = 5). All data shown were mean ± SD. # P
Figure Legend Snippet: Effects of rhein on TLRs, Akt, MAPK and NF-κB signaling pathways after IAV infection. The treatment of the blank control (BC), negative control (NC), positive control (PC), and rhein-treated group (rhein) was same with that of the “antioxidant assay”. After 48 h p.i., the cells were harvested. The expressions of TLR2, TLR3, and TLR4, the phosphorylations of Akt, ERK, p38, JNK MAPK, and the nuclear translocation of NF-κB p65 were determined by western blotting assay. The experiment was repeated five times (n = 5). All data shown were mean ± SD. # P

Techniques Used: Infection, Negative Control, Positive Control, Translocation Assay, Western Blot

4) Product Images from "Delineation of in vitro chondrogenesis of human synovial stem cells following preconditioning using decellularized matrix"

Article Title: Delineation of in vitro chondrogenesis of human synovial stem cells following preconditioning using decellularized matrix

Journal: Acta biomaterialia

doi: 10.1016/j.actbio.2015.04.001

MAPK signals were actively involved in the chondrogenesis of dECM expanded hSDSCs. Western blot was used to evaluate Erk1/2, p38, and Jnk signals in both phosphorylated and nonphosphorylated formats in hSDSCs during cell expansion (A) and chondrogenic
Figure Legend Snippet: MAPK signals were actively involved in the chondrogenesis of dECM expanded hSDSCs. Western blot was used to evaluate Erk1/2, p38, and Jnk signals in both phosphorylated and nonphosphorylated formats in hSDSCs during cell expansion (A) and chondrogenic

Techniques Used: Western Blot

Related Articles

Centrifugation:

Article Title: Neuroprotective effects of Scrophularia buergeriana extract against glutamate-induced toxicity in SH-SY5Y cells
Article Snippet: Protein extraction and western blot analysis The treated SH-SY5Y cells were lysed with radioimmunoprecipitation assay (RIPA) buffer [50 mM Tris-HCl pH 7.4, 150 mM NaCl, 1 mM ethylenediaminetetraacetic acid (EDTA), 1% Triton X-100, 1% sodium deoxycholate, 0.1% sodium dodecyl sulfate (SDS) and 1 mM PMSE] and 1% protease inhibitor cocktail (Roche, Basel, Switzerland), followed by centrifugation at 2,000 x g for 15 min at 4°C. .. After washing, the membranes were incubated at 4°C overnight with the following appropriate antibodies: SOD1 (1:1,000; cat. no. sc-515404), SOD2 (1:1,000; cat. no. sc-137254) (both from Santa Cruz Biotechnology, Santa Cruz, CA, USA), GPx-1 (1:1,000; cat. no. 3206), p-p38 (1:1,000; cat. no. 9211), p38 (1:1,000; cat. no. 8690), Bax (1:1,000; cat. no. 2772), Bcl-2 (1:1,000; cat. no. 3498), cleaved caspase-3 (1:1,000; cat. no. 9664), cleaved PARP (1:1,000; cat. no. 5625) and β-actin (1:2,000; cat. no. 8457) (all from Cell Signaling Technology, Danvers, MA, USA) washed 3 times with TBST, and incubated with horseradish peroxidase-conjugated secondary antibodies: goat anti-rabbit (1:1,000; cat. no. SA002), goat anti-mouse (1:1,000; cat. no. SA001) (both from genDEPOT, Katy, TX, USA) for 1 h at room temperature.

Synthesized:

Article Title: The nNOS-p38MAPK Pathway Is Mediated by NOS1AP during Neuronal Death
Article Snippet: Peptides were synthesized as described previously ( ) or obtained from GENECUST or GenicBio: “-GESV,” NH2 -YAGQWGESV-COOH; “-GASA,” NH2 -YAGQWGASA-COOH; “-ESDV” or “NR2B9,” NH2 -KLSSIESDV-COOH; “-EADA,” NH2 -KLSSIEADA-COOH; “L-TAT,” NH2 -GRKKRRQRRR-COOH; “L-TAT-NR2B9C,” NH2 -GRKKRRQRRR-KLSSIESDV-COOH; “L-TAT-GESV,” NH2 -GRKKRRQRRR-YAGQWGESV-COOH; “L-TAT-GESVamide,” NH2 -GRKKRRQRRR-YAGQWGESV-CONH2 ; “L-TAT-GASA,” NH2 -GRKKRRQRRRYAGQWGASA-COOH; “L-TAT-cp4GESV,” NH2 -GRKKRRQRRRGESVYAGQW-COOH; “L-TAT-revGESV,” NH2 -GRKKRRQRRRVSEGWQGAY-COOH; “D-TAT-GESV,” NH2-dRdRdRdQdRdRdKdKdRG-YAGQWGESV-COOH; and “D-TAT-GASA,” NH2-dRdRdRdQdRdRdKdKdRG-YAGQWGASA-COOH where TAT refers to the sequence from HIV-TAT protein conferring membrane translocation to cargo; L-TAT refers to TAT sequence composed of L-form amino acids; D-TAT refers to TAT sequence in reverse order (“retroinverso”) composed of D-form amino acids; and dR, dW, and dK refer to D-forms of the amino acids arginine, glutamine, and lysine. .. Mouse antibody against GFP(JL-8) was from Clontech; GST(B14), NOS1(A-11), and MKK3(30) from Santa Cruz Biotechnology; p38(L53F8 and D13E1) from Cell Signaling Technology; PRK1/PKN1 (49/PRK1) from BD Transduction Laboratories; NeuN(A60/MAB377) from Millipore; MAP2 (AP20) from Leinco; GFAP(G-A-5/G3893) from Sigma; and β-actin from Genscript.

Blocking Assay:

Article Title: Neuroprotective effects of Scrophularia buergeriana extract against glutamate-induced toxicity in SH-SY5Y cells
Article Snippet: The membranes were blocked with commercial blocking buffer (Thermo Fisher Scientific) and washed 3 times with Tris-buffered saline containing 0.1% Tween-20 (TBST). .. After washing, the membranes were incubated at 4°C overnight with the following appropriate antibodies: SOD1 (1:1,000; cat. no. sc-515404), SOD2 (1:1,000; cat. no. sc-137254) (both from Santa Cruz Biotechnology, Santa Cruz, CA, USA), GPx-1 (1:1,000; cat. no. 3206), p-p38 (1:1,000; cat. no. 9211), p38 (1:1,000; cat. no. 8690), Bax (1:1,000; cat. no. 2772), Bcl-2 (1:1,000; cat. no. 3498), cleaved caspase-3 (1:1,000; cat. no. 9664), cleaved PARP (1:1,000; cat. no. 5625) and β-actin (1:2,000; cat. no. 8457) (all from Cell Signaling Technology, Danvers, MA, USA) washed 3 times with TBST, and incubated with horseradish peroxidase-conjugated secondary antibodies: goat anti-rabbit (1:1,000; cat. no. SA002), goat anti-mouse (1:1,000; cat. no. SA001) (both from genDEPOT, Katy, TX, USA) for 1 h at room temperature.

Enzyme-linked Immunosorbent Assay:

Article Title: Involvement of NADPH oxidase 1 in UVB-induced cell signaling and cytotoxicity in human keratinocytes
Article Snippet: Paragraph title: Immunoblotting, ELISA assay, Immunofluorescence ... The supernatant (10,000 g, 10 min, 4 °C) was performed for immunoblotting with antibodies against phospho-Erk, Erk, phosphor-p38, p38, phospho-Akt and Akt (Cell Signaling Technology, 1:1000), or β-actin (Sigma, 1:2000).

Incubation:

Article Title: Neuroprotective effects of Scrophularia buergeriana extract against glutamate-induced toxicity in SH-SY5Y cells
Article Snippet: .. After washing, the membranes were incubated at 4°C overnight with the following appropriate antibodies: SOD1 (1:1,000; cat. no. sc-515404), SOD2 (1:1,000; cat. no. sc-137254) (both from Santa Cruz Biotechnology, Santa Cruz, CA, USA), GPx-1 (1:1,000; cat. no. 3206), p-p38 (1:1,000; cat. no. 9211), p38 (1:1,000; cat. no. 8690), Bax (1:1,000; cat. no. 2772), Bcl-2 (1:1,000; cat. no. 3498), cleaved caspase-3 (1:1,000; cat. no. 9664), cleaved PARP (1:1,000; cat. no. 5625) and β-actin (1:2,000; cat. no. 8457) (all from Cell Signaling Technology, Danvers, MA, USA) washed 3 times with TBST, and incubated with horseradish peroxidase-conjugated secondary antibodies: goat anti-rabbit (1:1,000; cat. no. SA002), goat anti-mouse (1:1,000; cat. no. SA001) (both from genDEPOT, Katy, TX, USA) for 1 h at room temperature. ..

Article Title: Delineation of in vitro chondrogenesis of human synovial stem cells following preconditioning using decellularized matrix
Article Snippet: The membrane was incubated with primary monoclonal antibodies in 5% bovine serum albumin (BSA) in TBST buffer (10 mM Tris-HCl, pH 7.5, 150 mM NaCl, 0.05% Tween-20) for 1 h (β-actin served as an internal control), followed by the secondary antibody of horseradish peroxidase–conjugated goat anti-mouse (Thermo Fisher Scientific) for 1 h. SuperSignal West Femto Maximum Sensitivity Substrate and CL-XPosure Film (Thermo Fisher Scientific) were used for exposure. .. The primary antibodies used in immunoblotting included the MAPK family antibody sampler kit [extracellular signal-regulated protein kinases 1 and 2 (Erk1/2), Jun N-terminal kinase (Jnk), and p38], phosphorylated (p-) MAPK family antibody sampler kit, and the Wnt signaling antibody sampler kit (Cell Signaling).

BIA-KA:

Article Title: Neuroprotective effects of Scrophularia buergeriana extract against glutamate-induced toxicity in SH-SY5Y cells
Article Snippet: The supernatant was collected, and the protein concentration was assessed using a bicinchoninic acid (BCA) protein assay kit (Thermo Fisher Scientific). .. After washing, the membranes were incubated at 4°C overnight with the following appropriate antibodies: SOD1 (1:1,000; cat. no. sc-515404), SOD2 (1:1,000; cat. no. sc-137254) (both from Santa Cruz Biotechnology, Santa Cruz, CA, USA), GPx-1 (1:1,000; cat. no. 3206), p-p38 (1:1,000; cat. no. 9211), p38 (1:1,000; cat. no. 8690), Bax (1:1,000; cat. no. 2772), Bcl-2 (1:1,000; cat. no. 3498), cleaved caspase-3 (1:1,000; cat. no. 9664), cleaved PARP (1:1,000; cat. no. 5625) and β-actin (1:2,000; cat. no. 8457) (all from Cell Signaling Technology, Danvers, MA, USA) washed 3 times with TBST, and incubated with horseradish peroxidase-conjugated secondary antibodies: goat anti-rabbit (1:1,000; cat. no. SA002), goat anti-mouse (1:1,000; cat. no. SA001) (both from genDEPOT, Katy, TX, USA) for 1 h at room temperature.

Article Title: Delineation of in vitro chondrogenesis of human synovial stem cells following preconditioning using decellularized matrix
Article Snippet: Total protein was quantified using the BCA™ Protein Assay Kit (Thermo Fisher Scientific). .. The primary antibodies used in immunoblotting included the MAPK family antibody sampler kit [extracellular signal-regulated protein kinases 1 and 2 (Erk1/2), Jun N-terminal kinase (Jnk), and p38], phosphorylated (p-) MAPK family antibody sampler kit, and the Wnt signaling antibody sampler kit (Cell Signaling).

Article Title: HIV Tat Induces Expression of ICAM-1 in HUVECs: Implications for miR-221/-222 in HIV-Associated Cardiomyopathy
Article Snippet: Western Blot Analysis Treated cells or tissue were lysed using the Mammalian Cell Lysis kit (Sigma, St. Louis, MO, USA) and the NE-PER Nuclear and Cytoplasmic Extraction kit (Pierce, Rockford, IL, USA) and quantified using the micro BCA Protein Assay kit (Pierce, Rockford, IL, USA). .. Western blots were then probed with antibodies recognizing the ICAM-1, ERK1/2, JNK, p38,NF-κB p65 (1∶200; Cell Signaling Technology, Danvers, MA, USA) and β-actin (1∶4000; Sigma, St. Louis, MO, USA).

Modification:

Article Title: Anti-influenza A virus activity of rhein through regulating oxidative stress, TLR4, Akt, MAPK, and NF-κB signal pathways
Article Snippet: EGF (# 8916), anisomycin (# 2222) and antibodies for human ERK1/2 (# 8867), p-ERK1/2 (# 13148), p-JNK (# 3708), JNK(# 4671), p-p38(# 4092), p38(# 14451), p65 (# 4764) and β-actin (# 12262) proteins were bought from Cell Signaling Technology® Inc Company (Danvers, MA, USA). .. Rhein was dissolved in DMSO as stock solution, and diluted with Dulbecco’s modified Eagle medium (DMEM, Invitrogen, Carlsbad, CA, USA) when used.

Western Blot:

Article Title: Neuroprotective effects of Scrophularia buergeriana extract against glutamate-induced toxicity in SH-SY5Y cells
Article Snippet: Paragraph title: Protein extraction and western blot analysis ... After washing, the membranes were incubated at 4°C overnight with the following appropriate antibodies: SOD1 (1:1,000; cat. no. sc-515404), SOD2 (1:1,000; cat. no. sc-137254) (both from Santa Cruz Biotechnology, Santa Cruz, CA, USA), GPx-1 (1:1,000; cat. no. 3206), p-p38 (1:1,000; cat. no. 9211), p38 (1:1,000; cat. no. 8690), Bax (1:1,000; cat. no. 2772), Bcl-2 (1:1,000; cat. no. 3498), cleaved caspase-3 (1:1,000; cat. no. 9664), cleaved PARP (1:1,000; cat. no. 5625) and β-actin (1:2,000; cat. no. 8457) (all from Cell Signaling Technology, Danvers, MA, USA) washed 3 times with TBST, and incubated with horseradish peroxidase-conjugated secondary antibodies: goat anti-rabbit (1:1,000; cat. no. SA002), goat anti-mouse (1:1,000; cat. no. SA001) (both from genDEPOT, Katy, TX, USA) for 1 h at room temperature.

Article Title: Delineation of in vitro chondrogenesis of human synovial stem cells following preconditioning using decellularized matrix
Article Snippet: Paragraph title: 2.8 Characterization of both MAPK and Wnt signals using Western blot ... The primary antibodies used in immunoblotting included the MAPK family antibody sampler kit [extracellular signal-regulated protein kinases 1 and 2 (Erk1/2), Jun N-terminal kinase (Jnk), and p38], phosphorylated (p-) MAPK family antibody sampler kit, and the Wnt signaling antibody sampler kit (Cell Signaling).

Article Title: HIV Tat Induces Expression of ICAM-1 in HUVECs: Implications for miR-221/-222 in HIV-Associated Cardiomyopathy
Article Snippet: .. Western blots were then probed with antibodies recognizing the ICAM-1, ERK1/2, JNK, p38,NF-κB p65 (1∶200; Cell Signaling Technology, Danvers, MA, USA) and β-actin (1∶4000; Sigma, St. Louis, MO, USA). ..

Translocation Assay:

Article Title: The nNOS-p38MAPK Pathway Is Mediated by NOS1AP during Neuronal Death
Article Snippet: Peptides were synthesized as described previously ( ) or obtained from GENECUST or GenicBio: “-GESV,” NH2 -YAGQWGESV-COOH; “-GASA,” NH2 -YAGQWGASA-COOH; “-ESDV” or “NR2B9,” NH2 -KLSSIESDV-COOH; “-EADA,” NH2 -KLSSIEADA-COOH; “L-TAT,” NH2 -GRKKRRQRRR-COOH; “L-TAT-NR2B9C,” NH2 -GRKKRRQRRR-KLSSIESDV-COOH; “L-TAT-GESV,” NH2 -GRKKRRQRRR-YAGQWGESV-COOH; “L-TAT-GESVamide,” NH2 -GRKKRRQRRR-YAGQWGESV-CONH2 ; “L-TAT-GASA,” NH2 -GRKKRRQRRRYAGQWGASA-COOH; “L-TAT-cp4GESV,” NH2 -GRKKRRQRRRGESVYAGQW-COOH; “L-TAT-revGESV,” NH2 -GRKKRRQRRRVSEGWQGAY-COOH; “D-TAT-GESV,” NH2-dRdRdRdQdRdRdKdKdRG-YAGQWGESV-COOH; and “D-TAT-GASA,” NH2-dRdRdRdQdRdRdKdKdRG-YAGQWGASA-COOH where TAT refers to the sequence from HIV-TAT protein conferring membrane translocation to cargo; L-TAT refers to TAT sequence composed of L-form amino acids; D-TAT refers to TAT sequence in reverse order (“retroinverso”) composed of D-form amino acids; and dR, dW, and dK refer to D-forms of the amino acids arginine, glutamine, and lysine. .. Mouse antibody against GFP(JL-8) was from Clontech; GST(B14), NOS1(A-11), and MKK3(30) from Santa Cruz Biotechnology; p38(L53F8 and D13E1) from Cell Signaling Technology; PRK1/PKN1 (49/PRK1) from BD Transduction Laboratories; NeuN(A60/MAB377) from Millipore; MAP2 (AP20) from Leinco; GFAP(G-A-5/G3893) from Sigma; and β-actin from Genscript.

Protease Inhibitor:

Article Title: Neuroprotective effects of Scrophularia buergeriana extract against glutamate-induced toxicity in SH-SY5Y cells
Article Snippet: Protein extraction and western blot analysis The treated SH-SY5Y cells were lysed with radioimmunoprecipitation assay (RIPA) buffer [50 mM Tris-HCl pH 7.4, 150 mM NaCl, 1 mM ethylenediaminetetraacetic acid (EDTA), 1% Triton X-100, 1% sodium deoxycholate, 0.1% sodium dodecyl sulfate (SDS) and 1 mM PMSE] and 1% protease inhibitor cocktail (Roche, Basel, Switzerland), followed by centrifugation at 2,000 x g for 15 min at 4°C. .. After washing, the membranes were incubated at 4°C overnight with the following appropriate antibodies: SOD1 (1:1,000; cat. no. sc-515404), SOD2 (1:1,000; cat. no. sc-137254) (both from Santa Cruz Biotechnology, Santa Cruz, CA, USA), GPx-1 (1:1,000; cat. no. 3206), p-p38 (1:1,000; cat. no. 9211), p38 (1:1,000; cat. no. 8690), Bax (1:1,000; cat. no. 2772), Bcl-2 (1:1,000; cat. no. 3498), cleaved caspase-3 (1:1,000; cat. no. 9664), cleaved PARP (1:1,000; cat. no. 5625) and β-actin (1:2,000; cat. no. 8457) (all from Cell Signaling Technology, Danvers, MA, USA) washed 3 times with TBST, and incubated with horseradish peroxidase-conjugated secondary antibodies: goat anti-rabbit (1:1,000; cat. no. SA002), goat anti-mouse (1:1,000; cat. no. SA001) (both from genDEPOT, Katy, TX, USA) for 1 h at room temperature.

Protein Concentration:

Article Title: Neuroprotective effects of Scrophularia buergeriana extract against glutamate-induced toxicity in SH-SY5Y cells
Article Snippet: The supernatant was collected, and the protein concentration was assessed using a bicinchoninic acid (BCA) protein assay kit (Thermo Fisher Scientific). .. After washing, the membranes were incubated at 4°C overnight with the following appropriate antibodies: SOD1 (1:1,000; cat. no. sc-515404), SOD2 (1:1,000; cat. no. sc-137254) (both from Santa Cruz Biotechnology, Santa Cruz, CA, USA), GPx-1 (1:1,000; cat. no. 3206), p-p38 (1:1,000; cat. no. 9211), p38 (1:1,000; cat. no. 8690), Bax (1:1,000; cat. no. 2772), Bcl-2 (1:1,000; cat. no. 3498), cleaved caspase-3 (1:1,000; cat. no. 9664), cleaved PARP (1:1,000; cat. no. 5625) and β-actin (1:2,000; cat. no. 8457) (all from Cell Signaling Technology, Danvers, MA, USA) washed 3 times with TBST, and incubated with horseradish peroxidase-conjugated secondary antibodies: goat anti-rabbit (1:1,000; cat. no. SA002), goat anti-mouse (1:1,000; cat. no. SA001) (both from genDEPOT, Katy, TX, USA) for 1 h at room temperature.

Sequencing:

Article Title: The nNOS-p38MAPK Pathway Is Mediated by NOS1AP during Neuronal Death
Article Snippet: Peptides were synthesized as described previously ( ) or obtained from GENECUST or GenicBio: “-GESV,” NH2 -YAGQWGESV-COOH; “-GASA,” NH2 -YAGQWGASA-COOH; “-ESDV” or “NR2B9,” NH2 -KLSSIESDV-COOH; “-EADA,” NH2 -KLSSIEADA-COOH; “L-TAT,” NH2 -GRKKRRQRRR-COOH; “L-TAT-NR2B9C,” NH2 -GRKKRRQRRR-KLSSIESDV-COOH; “L-TAT-GESV,” NH2 -GRKKRRQRRR-YAGQWGESV-COOH; “L-TAT-GESVamide,” NH2 -GRKKRRQRRR-YAGQWGESV-CONH2 ; “L-TAT-GASA,” NH2 -GRKKRRQRRRYAGQWGASA-COOH; “L-TAT-cp4GESV,” NH2 -GRKKRRQRRRGESVYAGQW-COOH; “L-TAT-revGESV,” NH2 -GRKKRRQRRRVSEGWQGAY-COOH; “D-TAT-GESV,” NH2-dRdRdRdQdRdRdKdKdRG-YAGQWGESV-COOH; and “D-TAT-GASA,” NH2-dRdRdRdQdRdRdKdKdRG-YAGQWGASA-COOH where TAT refers to the sequence from HIV-TAT protein conferring membrane translocation to cargo; L-TAT refers to TAT sequence composed of L-form amino acids; D-TAT refers to TAT sequence in reverse order (“retroinverso”) composed of D-form amino acids; and dR, dW, and dK refer to D-forms of the amino acids arginine, glutamine, and lysine. .. Mouse antibody against GFP(JL-8) was from Clontech; GST(B14), NOS1(A-11), and MKK3(30) from Santa Cruz Biotechnology; p38(L53F8 and D13E1) from Cell Signaling Technology; PRK1/PKN1 (49/PRK1) from BD Transduction Laboratories; NeuN(A60/MAB377) from Millipore; MAP2 (AP20) from Leinco; GFAP(G-A-5/G3893) from Sigma; and β-actin from Genscript.

Immunofluorescence:

Article Title: Involvement of NADPH oxidase 1 in UVB-induced cell signaling and cytotoxicity in human keratinocytes
Article Snippet: Paragraph title: Immunoblotting, ELISA assay, Immunofluorescence ... The supernatant (10,000 g, 10 min, 4 °C) was performed for immunoblotting with antibodies against phospho-Erk, Erk, phosphor-p38, p38, phospho-Akt and Akt (Cell Signaling Technology, 1:1000), or β-actin (Sigma, 1:2000).

Nucleic Acid Electrophoresis:

Article Title: Neuroprotective effects of Scrophularia buergeriana extract against glutamate-induced toxicity in SH-SY5Y cells
Article Snippet: Equal amounts of protein (20 µg/lane) were separated by 10-15% SDS-polyacrylamide gel electrophoresis and transferred onto polyvinylidene difluoride membranes (Bio-Rad, Hercules, CA, USA). .. After washing, the membranes were incubated at 4°C overnight with the following appropriate antibodies: SOD1 (1:1,000; cat. no. sc-515404), SOD2 (1:1,000; cat. no. sc-137254) (both from Santa Cruz Biotechnology, Santa Cruz, CA, USA), GPx-1 (1:1,000; cat. no. 3206), p-p38 (1:1,000; cat. no. 9211), p38 (1:1,000; cat. no. 8690), Bax (1:1,000; cat. no. 2772), Bcl-2 (1:1,000; cat. no. 3498), cleaved caspase-3 (1:1,000; cat. no. 9664), cleaved PARP (1:1,000; cat. no. 5625) and β-actin (1:2,000; cat. no. 8457) (all from Cell Signaling Technology, Danvers, MA, USA) washed 3 times with TBST, and incubated with horseradish peroxidase-conjugated secondary antibodies: goat anti-rabbit (1:1,000; cat. no. SA002), goat anti-mouse (1:1,000; cat. no. SA001) (both from genDEPOT, Katy, TX, USA) for 1 h at room temperature.

Protein Extraction:

Article Title: Neuroprotective effects of Scrophularia buergeriana extract against glutamate-induced toxicity in SH-SY5Y cells
Article Snippet: Paragraph title: Protein extraction and western blot analysis ... After washing, the membranes were incubated at 4°C overnight with the following appropriate antibodies: SOD1 (1:1,000; cat. no. sc-515404), SOD2 (1:1,000; cat. no. sc-137254) (both from Santa Cruz Biotechnology, Santa Cruz, CA, USA), GPx-1 (1:1,000; cat. no. 3206), p-p38 (1:1,000; cat. no. 9211), p38 (1:1,000; cat. no. 8690), Bax (1:1,000; cat. no. 2772), Bcl-2 (1:1,000; cat. no. 3498), cleaved caspase-3 (1:1,000; cat. no. 9664), cleaved PARP (1:1,000; cat. no. 5625) and β-actin (1:2,000; cat. no. 8457) (all from Cell Signaling Technology, Danvers, MA, USA) washed 3 times with TBST, and incubated with horseradish peroxidase-conjugated secondary antibodies: goat anti-rabbit (1:1,000; cat. no. SA002), goat anti-mouse (1:1,000; cat. no. SA001) (both from genDEPOT, Katy, TX, USA) for 1 h at room temperature.

Immunostaining:

Article Title: Involvement of NADPH oxidase 1 in UVB-induced cell signaling and cytotoxicity in human keratinocytes
Article Snippet: The supernatant (10,000 g, 10 min, 4 °C) was performed for immunoblotting with antibodies against phospho-Erk, Erk, phosphor-p38, p38, phospho-Akt and Akt (Cell Signaling Technology, 1:1000), or β-actin (Sigma, 1:2000). .. For Nox1 immunostaining, cells were fixed with 10% formalin, and permeabilized with 0.2% saponin, and immunostained with anti-Nox1 (Santa Cruz), followed by anti-rabbit secondary antibody (FITC, Sigma).

Software:

Article Title: Neuroprotective effects of Scrophularia buergeriana extract against glutamate-induced toxicity in SH-SY5Y cells
Article Snippet: After washing, the membranes were incubated at 4°C overnight with the following appropriate antibodies: SOD1 (1:1,000; cat. no. sc-515404), SOD2 (1:1,000; cat. no. sc-137254) (both from Santa Cruz Biotechnology, Santa Cruz, CA, USA), GPx-1 (1:1,000; cat. no. 3206), p-p38 (1:1,000; cat. no. 9211), p38 (1:1,000; cat. no. 8690), Bax (1:1,000; cat. no. 2772), Bcl-2 (1:1,000; cat. no. 3498), cleaved caspase-3 (1:1,000; cat. no. 9664), cleaved PARP (1:1,000; cat. no. 5625) and β-actin (1:2,000; cat. no. 8457) (all from Cell Signaling Technology, Danvers, MA, USA) washed 3 times with TBST, and incubated with horseradish peroxidase-conjugated secondary antibodies: goat anti-rabbit (1:1,000; cat. no. SA002), goat anti-mouse (1:1,000; cat. no. SA001) (both from genDEPOT, Katy, TX, USA) for 1 h at room temperature. .. Densitometry was performed using Image-Pro Plus software (6.0 version; Media Cybernetics, Inc., USA)

Article Title: Delineation of in vitro chondrogenesis of human synovial stem cells following preconditioning using decellularized matrix
Article Snippet: The primary antibodies used in immunoblotting included the MAPK family antibody sampler kit [extracellular signal-regulated protein kinases 1 and 2 (Erk1/2), Jun N-terminal kinase (Jnk), and p38], phosphorylated (p-) MAPK family antibody sampler kit, and the Wnt signaling antibody sampler kit (Cell Signaling). .. ImageJ software was used to quantify immunoblotting bands.

Article Title: HIV Tat Induces Expression of ICAM-1 in HUVECs: Implications for miR-221/-222 in HIV-Associated Cardiomyopathy
Article Snippet: Western blots were then probed with antibodies recognizing the ICAM-1, ERK1/2, JNK, p38,NF-κB p65 (1∶200; Cell Signaling Technology, Danvers, MA, USA) and β-actin (1∶4000; Sigma, St. Louis, MO, USA). .. Signals were detected by chemiluminescence and imaged on the FLA-5100 (Fujifilm, Valhalla, NY, USA) digital image scanner; densitometry was performed utilizing Image J software (NIH) .

Radio Immunoprecipitation:

Article Title: Neuroprotective effects of Scrophularia buergeriana extract against glutamate-induced toxicity in SH-SY5Y cells
Article Snippet: Protein extraction and western blot analysis The treated SH-SY5Y cells were lysed with radioimmunoprecipitation assay (RIPA) buffer [50 mM Tris-HCl pH 7.4, 150 mM NaCl, 1 mM ethylenediaminetetraacetic acid (EDTA), 1% Triton X-100, 1% sodium deoxycholate, 0.1% sodium dodecyl sulfate (SDS) and 1 mM PMSE] and 1% protease inhibitor cocktail (Roche, Basel, Switzerland), followed by centrifugation at 2,000 x g for 15 min at 4°C. .. After washing, the membranes were incubated at 4°C overnight with the following appropriate antibodies: SOD1 (1:1,000; cat. no. sc-515404), SOD2 (1:1,000; cat. no. sc-137254) (both from Santa Cruz Biotechnology, Santa Cruz, CA, USA), GPx-1 (1:1,000; cat. no. 3206), p-p38 (1:1,000; cat. no. 9211), p38 (1:1,000; cat. no. 8690), Bax (1:1,000; cat. no. 2772), Bcl-2 (1:1,000; cat. no. 3498), cleaved caspase-3 (1:1,000; cat. no. 9664), cleaved PARP (1:1,000; cat. no. 5625) and β-actin (1:2,000; cat. no. 8457) (all from Cell Signaling Technology, Danvers, MA, USA) washed 3 times with TBST, and incubated with horseradish peroxidase-conjugated secondary antibodies: goat anti-rabbit (1:1,000; cat. no. SA002), goat anti-mouse (1:1,000; cat. no. SA001) (both from genDEPOT, Katy, TX, USA) for 1 h at room temperature.

Sulforhodamine B Assay:

Article Title: Anti-influenza A virus activity of rhein through regulating oxidative stress, TLR4, Akt, MAPK, and NF-κB signal pathways
Article Snippet: DMSO, Tosylsulfonyl phenylalanyl chloromethyl ketone (TPCK)- trypsin (# 4370285-1KT), ribavirin (# R9644-10MG) and sulforhodamine B (SRB, # 230162-5G) were purchased from Sigma-Aldrich, Inc (St. Louis, MO, USA). .. EGF (# 8916), anisomycin (# 2222) and antibodies for human ERK1/2 (# 8867), p-ERK1/2 (# 13148), p-JNK (# 3708), JNK(# 4671), p-p38(# 4092), p38(# 14451), p65 (# 4764) and β-actin (# 12262) proteins were bought from Cell Signaling Technology® Inc Company (Danvers, MA, USA).

Laser Capture Microdissection:

Article Title: Anti-influenza A virus activity of rhein through regulating oxidative stress, TLR4, Akt, MAPK, and NF-κB signal pathways
Article Snippet: LAM-MS (# tlrl-lams), Poly(I:C) (# tlrl-pic), LPS-B5 (# tlrl-pb5lps) were purchased from InvivoGen (San Diego, California, USA). .. EGF (# 8916), anisomycin (# 2222) and antibodies for human ERK1/2 (# 8867), p-ERK1/2 (# 13148), p-JNK (# 3708), JNK(# 4671), p-p38(# 4092), p38(# 14451), p65 (# 4764) and β-actin (# 12262) proteins were bought from Cell Signaling Technology® Inc Company (Danvers, MA, USA).

Immunoprecipitation:

Article Title: The nNOS-p38MAPK Pathway Is Mediated by NOS1AP during Neuronal Death
Article Snippet: Mouse antibody against GFP(JL-8) was from Clontech; GST(B14), NOS1(A-11), and MKK3(30) from Santa Cruz Biotechnology; p38(L53F8 and D13E1) from Cell Signaling Technology; PRK1/PKN1 (49/PRK1) from BD Transduction Laboratories; NeuN(A60/MAB377) from Millipore; MAP2 (AP20) from Leinco; GFAP(G-A-5/G3893) from Sigma; and β-actin from Genscript. .. This antiserum selectively immunoprecipitated MKK3 not MKK6 (our unpublished observations).

Lysis:

Article Title: Delineation of in vitro chondrogenesis of human synovial stem cells following preconditioning using decellularized matrix
Article Snippet: Adult hSDSCs from cell expansion (day 1, 3, and 5) and pellets before (day 0) and after (day 1 and 7) chondrogenic induction were dissolved in the lysis buffer (Cell Signaling, Boston, MA) with protease inhibitors. .. The primary antibodies used in immunoblotting included the MAPK family antibody sampler kit [extracellular signal-regulated protein kinases 1 and 2 (Erk1/2), Jun N-terminal kinase (Jnk), and p38], phosphorylated (p-) MAPK family antibody sampler kit, and the Wnt signaling antibody sampler kit (Cell Signaling).

Article Title: HIV Tat Induces Expression of ICAM-1 in HUVECs: Implications for miR-221/-222 in HIV-Associated Cardiomyopathy
Article Snippet: Western Blot Analysis Treated cells or tissue were lysed using the Mammalian Cell Lysis kit (Sigma, St. Louis, MO, USA) and the NE-PER Nuclear and Cytoplasmic Extraction kit (Pierce, Rockford, IL, USA) and quantified using the micro BCA Protein Assay kit (Pierce, Rockford, IL, USA). .. Western blots were then probed with antibodies recognizing the ICAM-1, ERK1/2, JNK, p38,NF-κB p65 (1∶200; Cell Signaling Technology, Danvers, MA, USA) and β-actin (1∶4000; Sigma, St. Louis, MO, USA).

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    Cell Signaling Technology Inc p38
    Effect of <t>p38,</t> JNK1/2 and PI3K inhibition on cell proliferation of LLC-PK1 cells treated with telocinobufagin (TCB). Serum-starved LLC-PK1 cells were treated with 100 nM TCB in 2.5% FBS for 72 h with or without 10 μM SB202180 (p38 inhibitor) ( a ), 1.5 μM SB600125 (JNK1/2 inhibitor) ( b ), or 5 μM LY294002 (PI3K inhibitor) ( c ). Trypan blue-free viable cells were counted in Neubauer chamber. Data are the mean ± SEM of at least three independent experiments in duplicate. *** p
    P38, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 99/100, based on 342 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Effect of p38, JNK1/2 and PI3K inhibition on cell proliferation of LLC-PK1 cells treated with telocinobufagin (TCB). Serum-starved LLC-PK1 cells were treated with 100 nM TCB in 2.5% FBS for 72 h with or without 10 μM SB202180 (p38 inhibitor) ( a ), 1.5 μM SB600125 (JNK1/2 inhibitor) ( b ), or 5 μM LY294002 (PI3K inhibitor) ( c ). Trypan blue-free viable cells were counted in Neubauer chamber. Data are the mean ± SEM of at least three independent experiments in duplicate. *** p

    Journal: International Journal of Molecular Sciences

    Article Title: Telocinobufagin and Marinobufagin Produce Different Effects in LLC-PK1 Cells: A Case of Functional Selectivity of Bufadienolides

    doi: 10.3390/ijms19092769

    Figure Lengend Snippet: Effect of p38, JNK1/2 and PI3K inhibition on cell proliferation of LLC-PK1 cells treated with telocinobufagin (TCB). Serum-starved LLC-PK1 cells were treated with 100 nM TCB in 2.5% FBS for 72 h with or without 10 μM SB202180 (p38 inhibitor) ( a ), 1.5 μM SB600125 (JNK1/2 inhibitor) ( b ), or 5 μM LY294002 (PI3K inhibitor) ( c ). Trypan blue-free viable cells were counted in Neubauer chamber. Data are the mean ± SEM of at least three independent experiments in duplicate. *** p

    Article Snippet: Cell Counting Assay In 24-well plates, 1–2 × 104 LLC-PK1 cells/well were treated with 1, 10 or 100 nM CTS for 24, 48, and 72 h. In order to investigate the involvement of intracellular signaling pathways, 100 nM CTS were incubated with or without inhibitors of Src (SU6656, 10 μM; Sigma-Aldrich, St. Louis, MO, USA), ERK1/2-MEK1/2 (U0126, 10 μM; Cell Signaling Technology, Danvers, MA, USA), PI3K (LY294002, 5 μM; Cell Signaling Technology, Danvers, MA, USA), p38 (SB202190, 10 μM; Cell Signaling Technology, Danvers, MA, USA), JNK1/2 (SP600125, 1.5 μM; Cell Signaling Technology, Danvers, MA, USA), or GSK-3β (BIO, 0.5, 1, and 5 μM; Sigma-Aldrich, St. Louis, MO, USA) for 72 h. For MβCD (Sigma-Aldrich, St. Louis, MO, USA) assay, cells were pretreated with 10 mM MβCD for 30 min and then 1 mM MβCD + 100 nM TCB in 2.5% FBS for 72 h. At these time points, two wells from each group were trypsinized and the number of Trypan blue-viable cells was counted in Neubauer chamber (hemocytometer).

    Techniques: Inhibition

    Blockade of 4-1BB signaling attenuates the secretion of pro-fibrotic mediators by MH-S cells. Transfected (Len-cont. and sh-4-1BB) MH-S cells were treated with or without NQDI 1 (10 µM), 4-1BBIg (10 µg/mL), or IgG1 (10 µg/mL) for 2 h, then exposed to crystalline silica (50 µg/cm 2 ) for 12 h. (A) Western blots analysis of ASK-1 and downstream mitogen-activated protein kinase proteins (p38 and JNK/stress activated protein kinase) and their phosphorylated forms. (B–D) Levels of phospho-ASK1, phospho-p38, and phospho-JNK were normalized to those of β-actin ( n = 3). (E,G) Western blots analysis of IκBα and phospho-IκBα. (F,H) The level of phospho-IκBα was normalized to those of β-actin ( n = 3). (I–K) The expressions of MMP12, MMP9, and monocyte chemoattractant protein-1 were detected by real-time polymerase chain reaction analysis ( n = 4). ELISA analysis of cytokines in the culture supernatants. (L) IL-1β, (M) IL-6, (N) tumor necrosis factor-α ( n = 4). Data were shown as mean ± SEM (* p ≤ 0.05; ** p ≤ 0.01; *** p ≤ 0.001; ns, not significant). The data were representative of three independent experiments.

    Journal: Frontiers in Immunology

    Article Title: 4-1BB Signaling Promotes Alveolar Macrophages-Mediated Pro-Fibrotic Responses and Crystalline Silica-Induced Pulmonary Fibrosis in Mice

    doi: 10.3389/fimmu.2018.01848

    Figure Lengend Snippet: Blockade of 4-1BB signaling attenuates the secretion of pro-fibrotic mediators by MH-S cells. Transfected (Len-cont. and sh-4-1BB) MH-S cells were treated with or without NQDI 1 (10 µM), 4-1BBIg (10 µg/mL), or IgG1 (10 µg/mL) for 2 h, then exposed to crystalline silica (50 µg/cm 2 ) for 12 h. (A) Western blots analysis of ASK-1 and downstream mitogen-activated protein kinase proteins (p38 and JNK/stress activated protein kinase) and their phosphorylated forms. (B–D) Levels of phospho-ASK1, phospho-p38, and phospho-JNK were normalized to those of β-actin ( n = 3). (E,G) Western blots analysis of IκBα and phospho-IκBα. (F,H) The level of phospho-IκBα was normalized to those of β-actin ( n = 3). (I–K) The expressions of MMP12, MMP9, and monocyte chemoattractant protein-1 were detected by real-time polymerase chain reaction analysis ( n = 4). ELISA analysis of cytokines in the culture supernatants. (L) IL-1β, (M) IL-6, (N) tumor necrosis factor-α ( n = 4). Data were shown as mean ± SEM (* p ≤ 0.05; ** p ≤ 0.01; *** p ≤ 0.001; ns, not significant). The data were representative of three independent experiments.

    Article Snippet: The expression of CD137 (1:500, rat, Abcam, Cambridge, UK), ASK1 (1:2,000, rabbit, Abcam), phospho-ASK1 [1:500, rabbit, Cell Signaling Technology (CST), Danvers, MA, USA], p38 (1:1,000, rabbit, CST), phospho-p38 (1:1,000, rabbit, CST), JNK (1:1,000, rabbit, CST), phospho-JNK (1:1,000, rabbit, CST), IκBα (1:1,000, rabbit, CST), phospho-IκBα (1:1,000, rabbit, CST), MMP9 (1:1,000, rabbit, Abcam), MMP12 (1:2,000, rabbit, Abcam), and β-actin (1:1,000, rabbit, CST) in MH-S cells or lung tissues was measured by western blot.

    Techniques: Transfection, Western Blot, Real-time Polymerase Chain Reaction, Enzyme-linked Immunosorbent Assay

    Activation of 4-1BB signaling promotes the secretion of pro-fibrotic mediators by MH-S cells. MH-S cells treated with or without agonist 4-1BB mAb (10 µg/mL) or IgG (10 µg/mL) for 2 h prior to exposure to crystalline silica (50 µg/cm 2 ) for 12 h. (A) Western blots analysis of ASK-1 and downstream mitogen-activated protein kinase proteins (p38 and JNK/stress activated protein kinase) and their phosphorylated forms. (B–D) The levels of phospho-ASK1, phospho-p38, and phospho-JNK were normalized to those of β-actin ( n = 3). (E) Western blots analysis of IκBα and phospho-IκBα. (F) The level of phospho-IκBα was normalized to those of β-actin ( n = 3). (G–I) Real-time polymerase chain reaction analysis of MMP12, MMP9, and monocyte chemoattractant protein-1 mRNA expression ( n = 4). (J–L) ELISA analysis was used to quantify the secretion of IL-1β, IL-6 and tumor necrosis factor-α ( n = 4). The results were representative of three independent experiments. Results were graphed as the mean ± SEM (* p ≤ 0.05; ** p ≤ 0.01; *** p ≤ 0.001).

    Journal: Frontiers in Immunology

    Article Title: 4-1BB Signaling Promotes Alveolar Macrophages-Mediated Pro-Fibrotic Responses and Crystalline Silica-Induced Pulmonary Fibrosis in Mice

    doi: 10.3389/fimmu.2018.01848

    Figure Lengend Snippet: Activation of 4-1BB signaling promotes the secretion of pro-fibrotic mediators by MH-S cells. MH-S cells treated with or without agonist 4-1BB mAb (10 µg/mL) or IgG (10 µg/mL) for 2 h prior to exposure to crystalline silica (50 µg/cm 2 ) for 12 h. (A) Western blots analysis of ASK-1 and downstream mitogen-activated protein kinase proteins (p38 and JNK/stress activated protein kinase) and their phosphorylated forms. (B–D) The levels of phospho-ASK1, phospho-p38, and phospho-JNK were normalized to those of β-actin ( n = 3). (E) Western blots analysis of IκBα and phospho-IκBα. (F) The level of phospho-IκBα was normalized to those of β-actin ( n = 3). (G–I) Real-time polymerase chain reaction analysis of MMP12, MMP9, and monocyte chemoattractant protein-1 mRNA expression ( n = 4). (J–L) ELISA analysis was used to quantify the secretion of IL-1β, IL-6 and tumor necrosis factor-α ( n = 4). The results were representative of three independent experiments. Results were graphed as the mean ± SEM (* p ≤ 0.05; ** p ≤ 0.01; *** p ≤ 0.001).

    Article Snippet: The expression of CD137 (1:500, rat, Abcam, Cambridge, UK), ASK1 (1:2,000, rabbit, Abcam), phospho-ASK1 [1:500, rabbit, Cell Signaling Technology (CST), Danvers, MA, USA], p38 (1:1,000, rabbit, CST), phospho-p38 (1:1,000, rabbit, CST), JNK (1:1,000, rabbit, CST), phospho-JNK (1:1,000, rabbit, CST), IκBα (1:1,000, rabbit, CST), phospho-IκBα (1:1,000, rabbit, CST), MMP9 (1:1,000, rabbit, Abcam), MMP12 (1:2,000, rabbit, Abcam), and β-actin (1:1,000, rabbit, CST) in MH-S cells or lung tissues was measured by western blot.

    Techniques: Activation Assay, Western Blot, Real-time Polymerase Chain Reaction, Expressing, Enzyme-linked Immunosorbent Assay

    Dose‐dependent reduction of age‐related cardiac inflammation by phenolic compounds. (a) Representative microphotographs of left ventricular sections of the heart stained with hematoxylin‐eosin showing young, SHAM, DMSO, PC 2.5, PC 5, PC 10, and PC 20 groups. (b) Histograms showing cardiomyocytes width (μm) and semiquantitative scores of inflammation ( n = 6–8 per group). (c) CRP and IL‐6 plasma concentrations in all groups ( n = 6–8 per group). (d) Representative western blot analyses for p38 in rat cardiac tissue, normalized to GAPDH (in arbitrary units, a.u.) ( n = 3 for each protein and condition). * p

    Journal: Aging Cell

    Article Title: Long‐term intake of phenolic compounds attenuates age‐related cardiac remodeling, et al. Long‐term intake of phenolic compounds attenuates age‐related cardiac remodeling

    doi: 10.1111/acel.12894

    Figure Lengend Snippet: Dose‐dependent reduction of age‐related cardiac inflammation by phenolic compounds. (a) Representative microphotographs of left ventricular sections of the heart stained with hematoxylin‐eosin showing young, SHAM, DMSO, PC 2.5, PC 5, PC 10, and PC 20 groups. (b) Histograms showing cardiomyocytes width (μm) and semiquantitative scores of inflammation ( n = 6–8 per group). (c) CRP and IL‐6 plasma concentrations in all groups ( n = 6–8 per group). (d) Representative western blot analyses for p38 in rat cardiac tissue, normalized to GAPDH (in arbitrary units, a.u.) ( n = 3 for each protein and condition). * p

    Article Snippet: The blots were then incubated overnight with gentle agitation at 4°C with primary antibodies: NFATc3 (Santa‐Cruz Biotechnology, Dallas, TX, USA; 1/500), phospho‐Ser165 NFATc3 (p‐NFATc3) (Abcam; 1/500), PP2B‐Aβ (C‐20) (Santa‐Cruz Biotechnology; 1/1,000), SOD1 (Abcam; 1/2000), SOD2 (Abcam; 1/4,000), Smad2 (Abcam; 1/500), phospho‐S467 Smad2 (p‐Smad2) (Abcam; 1/500), Smad3 (Abcam; 1/1,000), phospho‐S423+S425 Smad3 (p‐Smad3) (Abcam; 1/1,000), pan‐CAMKII (Cell Signaling Technology, MA, USA; 1/1,000), pan‐phospho CAMKII (Cell Signaling Technology; 1/1,000), troponin I (C‐4) (Santa‐Cruz Biotechnology; 1/500), GSK 3β (Cell Signaling Technology; 1/1,000), phospho‐GSK 3β (Cell Signaling Technology; 1/1,000), ERK1/2 (Abcam; 1/1,000), phospho‐ERK 1/2 (Abcam; 1/10,000), P38 (Cell Signaling Technology; 1/1,000), phospho‐Thr180/Tyr182 P38 (Cell Signaling Technology; 1/1,000), NF‐ƙB p65 (Ser536) (Cell Signaling Technology; 1/1,000), phospho‐NF‐ƙB p65 (Ser536) (93H1) (Cell Signaling Technology; 1/1,000), and glyceraldehyde‐3‐phosphate dehydrogenase (GAPDH) (Abcam; 1/2,500 for all).

    Techniques: Staining, Western Blot

    Immunoactivity of phosphorylated p38 in IgA nephropathy patients according to the grade of interstitial fibrosis (original magnification x100). Control panel shows the phosphorylated p38 kidney tissue activity among patients who have no pathologic changes in kidney biopsy specimens. (Fig 1-A) The immunoactivity of phosphorylated p38 in tissues from IgA nephropathy patients with moderate interstitial fibrosis (Fig 1-D) was higher than that in tissues from patients with no interstitial fibrosis (Fig 1-B) or mild interstitial fibrosis (Fig 1-C). The immunoactivity of phosphorylated p38 in patients with a high degree of interstitial fibrosis was higher than immunoactivity in those with a low degree of kidney interstitial fibrosis (Fig 1-E; overall P

    Journal: PLoS ONE

    Article Title: p38 MAPK activity is associated with the histological degree of interstitial fibrosis in IgA nephropathy patients

    doi: 10.1371/journal.pone.0213981

    Figure Lengend Snippet: Immunoactivity of phosphorylated p38 in IgA nephropathy patients according to the grade of interstitial fibrosis (original magnification x100). Control panel shows the phosphorylated p38 kidney tissue activity among patients who have no pathologic changes in kidney biopsy specimens. (Fig 1-A) The immunoactivity of phosphorylated p38 in tissues from IgA nephropathy patients with moderate interstitial fibrosis (Fig 1-D) was higher than that in tissues from patients with no interstitial fibrosis (Fig 1-B) or mild interstitial fibrosis (Fig 1-C). The immunoactivity of phosphorylated p38 in patients with a high degree of interstitial fibrosis was higher than immunoactivity in those with a low degree of kidney interstitial fibrosis (Fig 1-E; overall P

    Article Snippet: In the groups with treated with 5 mg/kg/day and 10 mg/kg/day p38 MAPK inhibitor, the level of phosphorylated p38 showed a tendency to decrease compared with that in the vehicle group ( , P = 0.20 vehicle group vs. p38 MAPK inhibitor 5 mg/kg/day group).

    Techniques: Activity Assay

    Cellular mRNA expression of fibrosis-related markers after exposure to TGF-β in the presence of p38 MAPK inhibitor. (A) COL1 expression was increased after exposure to TGF-β (2 ng/ml). Treatment with the p38 MAPK inhibitor significantly reduced the cellular expression of COL1. (B) The decrease in the expression of fibronectin was not significant after concomitant exposure to TGF-β and p38 MAPK inhibitor. (C) Expression of periostin was increased after exposure to TGF-β (2 ng/ml). Treatment with p38 MAPK inhibitor reduced TGF-β-induced periostin expression. (n = 6 per group for each experiment, and in vitro experiments were repeated 3 times to confirm reproducibility) *, P

    Journal: PLoS ONE

    Article Title: p38 MAPK activity is associated with the histological degree of interstitial fibrosis in IgA nephropathy patients

    doi: 10.1371/journal.pone.0213981

    Figure Lengend Snippet: Cellular mRNA expression of fibrosis-related markers after exposure to TGF-β in the presence of p38 MAPK inhibitor. (A) COL1 expression was increased after exposure to TGF-β (2 ng/ml). Treatment with the p38 MAPK inhibitor significantly reduced the cellular expression of COL1. (B) The decrease in the expression of fibronectin was not significant after concomitant exposure to TGF-β and p38 MAPK inhibitor. (C) Expression of periostin was increased after exposure to TGF-β (2 ng/ml). Treatment with p38 MAPK inhibitor reduced TGF-β-induced periostin expression. (n = 6 per group for each experiment, and in vitro experiments were repeated 3 times to confirm reproducibility) *, P

    Article Snippet: In the groups with treated with 5 mg/kg/day and 10 mg/kg/day p38 MAPK inhibitor, the level of phosphorylated p38 showed a tendency to decrease compared with that in the vehicle group ( , P = 0.20 vehicle group vs. p38 MAPK inhibitor 5 mg/kg/day group).

    Techniques: Expressing, In Vitro

    Immunohistochemical staining showing the expression of phosphorylated p38 in the mouse renal tubulointerstitial area after UUO and administration of p38 MAPK inhibitor. Scale bar = 50 μm (original magnification x300). (A) Sham-operated group. (B) UUO-operated group administered 20% DMSO vehicle. (C) UUO-operated group administered 5 mg/kg/day p38 MAPK inhibitor. (D) UUO-operated group administered 10 mg/kg/day p38 MAPK inhibitor. Tubulointerstitial expression of phosphorylated p38 decreased significantly in the groups treated with p38 MAPK inhibitor. UUO, unilateral ureteral obstruction.

    Journal: PLoS ONE

    Article Title: p38 MAPK activity is associated with the histological degree of interstitial fibrosis in IgA nephropathy patients

    doi: 10.1371/journal.pone.0213981

    Figure Lengend Snippet: Immunohistochemical staining showing the expression of phosphorylated p38 in the mouse renal tubulointerstitial area after UUO and administration of p38 MAPK inhibitor. Scale bar = 50 μm (original magnification x300). (A) Sham-operated group. (B) UUO-operated group administered 20% DMSO vehicle. (C) UUO-operated group administered 5 mg/kg/day p38 MAPK inhibitor. (D) UUO-operated group administered 10 mg/kg/day p38 MAPK inhibitor. Tubulointerstitial expression of phosphorylated p38 decreased significantly in the groups treated with p38 MAPK inhibitor. UUO, unilateral ureteral obstruction.

    Article Snippet: In the groups with treated with 5 mg/kg/day and 10 mg/kg/day p38 MAPK inhibitor, the level of phosphorylated p38 showed a tendency to decrease compared with that in the vehicle group ( , P = 0.20 vehicle group vs. p38 MAPK inhibitor 5 mg/kg/day group).

    Techniques: Immunohistochemistry, Staining, Expressing

    Microscopic morphological changes in hTECs after treatment with p38 MAPK inhibitor. (A) Control hTECs show a normal ellipsoid shape and clear contour. (B) Exposure to TGF-β (2 ng/ml) induced bursting and speckled morphological changes in hTECs. (C) Exposure to TGF-β (2 ng/ml) in the presence of p38 MAPK inhibitor (2 μM). (D) Exposure to TGF-β (2 ng/ml) in the presence of p38 MAPK inhibitor (4 μM).

    Journal: PLoS ONE

    Article Title: p38 MAPK activity is associated with the histological degree of interstitial fibrosis in IgA nephropathy patients

    doi: 10.1371/journal.pone.0213981

    Figure Lengend Snippet: Microscopic morphological changes in hTECs after treatment with p38 MAPK inhibitor. (A) Control hTECs show a normal ellipsoid shape and clear contour. (B) Exposure to TGF-β (2 ng/ml) induced bursting and speckled morphological changes in hTECs. (C) Exposure to TGF-β (2 ng/ml) in the presence of p38 MAPK inhibitor (2 μM). (D) Exposure to TGF-β (2 ng/ml) in the presence of p38 MAPK inhibitor (4 μM).

    Article Snippet: In the groups with treated with 5 mg/kg/day and 10 mg/kg/day p38 MAPK inhibitor, the level of phosphorylated p38 showed a tendency to decrease compared with that in the vehicle group ( , P = 0.20 vehicle group vs. p38 MAPK inhibitor 5 mg/kg/day group).

    Techniques:

    Fibrosis-related renal protein expression as assessed with Western blot. (A) Expression of αSMA did not decrease after p38 MAPK inhibitor administration. (B) Expression of COL1 decreased significantly after administration of 10 mg/kg/day p38 MAPK inhibitor. (C) Expression of phosphorylated p38 MAPK decreased significantly after administration of 10 mg/kg/day p38 MAPK inhibitor. αSMA, α-smooth muscle actin; COL1, type 1 collagen. † , P

    Journal: PLoS ONE

    Article Title: p38 MAPK activity is associated with the histological degree of interstitial fibrosis in IgA nephropathy patients

    doi: 10.1371/journal.pone.0213981

    Figure Lengend Snippet: Fibrosis-related renal protein expression as assessed with Western blot. (A) Expression of αSMA did not decrease after p38 MAPK inhibitor administration. (B) Expression of COL1 decreased significantly after administration of 10 mg/kg/day p38 MAPK inhibitor. (C) Expression of phosphorylated p38 MAPK decreased significantly after administration of 10 mg/kg/day p38 MAPK inhibitor. αSMA, α-smooth muscle actin; COL1, type 1 collagen. † , P

    Article Snippet: In the groups with treated with 5 mg/kg/day and 10 mg/kg/day p38 MAPK inhibitor, the level of phosphorylated p38 showed a tendency to decrease compared with that in the vehicle group ( , P = 0.20 vehicle group vs. p38 MAPK inhibitor 5 mg/kg/day group).

    Techniques: Expressing, Western Blot

    Histologic Masson's trichrome stain findings of renal fibrosis after UUO and administration of a p38 MAPK inhibitor. Scale bar = 100 μm (original magnification x100). Mice from the sham-operated control group (A) show sparse tubulointerstitial fibrosis. UUO-operated group with administered 20% DMSO vehicle (B) shows prominent tubulointerstitial fibrosis. UUO-operated group administered p38 MAPK inhibitor at 5 mg/kg/day (C). UUO-operated group administered p38 MAPK inhibitor at 10 mg/kg/day (D). The extent of tubulointerstitial fibrosis in the groups administered p38 MAPK inhibitor was lower than that in the vehicle group (E). *, P

    Journal: PLoS ONE

    Article Title: p38 MAPK activity is associated with the histological degree of interstitial fibrosis in IgA nephropathy patients

    doi: 10.1371/journal.pone.0213981

    Figure Lengend Snippet: Histologic Masson's trichrome stain findings of renal fibrosis after UUO and administration of a p38 MAPK inhibitor. Scale bar = 100 μm (original magnification x100). Mice from the sham-operated control group (A) show sparse tubulointerstitial fibrosis. UUO-operated group with administered 20% DMSO vehicle (B) shows prominent tubulointerstitial fibrosis. UUO-operated group administered p38 MAPK inhibitor at 5 mg/kg/day (C). UUO-operated group administered p38 MAPK inhibitor at 10 mg/kg/day (D). The extent of tubulointerstitial fibrosis in the groups administered p38 MAPK inhibitor was lower than that in the vehicle group (E). *, P

    Article Snippet: In the groups with treated with 5 mg/kg/day and 10 mg/kg/day p38 MAPK inhibitor, the level of phosphorylated p38 showed a tendency to decrease compared with that in the vehicle group ( , P = 0.20 vehicle group vs. p38 MAPK inhibitor 5 mg/kg/day group).

    Techniques: Staining, Mouse Assay

    Fibrosis-related mRNA expression as assessed with RT-PCR. (A) Expression of αSMA was decreased in the 10 mg/kg/day p38 MAPK inhibitor group. (B) Expression of COL1 was decreased in the 10 mg/kg/day p38 MAPK inhibitor group. (C) Expression of fibronectin was decreased in the 5 and 10 mg/kg/day p38 MAPK inhibitor groups, with a dose-dependent pattern. (D) Expression of p38 MAPK was decreased in the 10 mg/kg/day p38 MAPK inhibitor group. (E) Expression of MCP-1 was decreased in the 10 mg/kg/day p38 MAPK inhibitor group. (F) The decrease in periostin expression was not statistically significant. αSMA, α-smooth muscle actin; COL1, type 1 collagen; MCP-1, monocyte chemoattractant protein-1; UUO, unilateral ureteral obstruction. *, P

    Journal: PLoS ONE

    Article Title: p38 MAPK activity is associated with the histological degree of interstitial fibrosis in IgA nephropathy patients

    doi: 10.1371/journal.pone.0213981

    Figure Lengend Snippet: Fibrosis-related mRNA expression as assessed with RT-PCR. (A) Expression of αSMA was decreased in the 10 mg/kg/day p38 MAPK inhibitor group. (B) Expression of COL1 was decreased in the 10 mg/kg/day p38 MAPK inhibitor group. (C) Expression of fibronectin was decreased in the 5 and 10 mg/kg/day p38 MAPK inhibitor groups, with a dose-dependent pattern. (D) Expression of p38 MAPK was decreased in the 10 mg/kg/day p38 MAPK inhibitor group. (E) Expression of MCP-1 was decreased in the 10 mg/kg/day p38 MAPK inhibitor group. (F) The decrease in periostin expression was not statistically significant. αSMA, α-smooth muscle actin; COL1, type 1 collagen; MCP-1, monocyte chemoattractant protein-1; UUO, unilateral ureteral obstruction. *, P

    Article Snippet: In the groups with treated with 5 mg/kg/day and 10 mg/kg/day p38 MAPK inhibitor, the level of phosphorylated p38 showed a tendency to decrease compared with that in the vehicle group ( , P = 0.20 vehicle group vs. p38 MAPK inhibitor 5 mg/kg/day group).

    Techniques: Expressing, Reverse Transcription Polymerase Chain Reaction