phosphorylated p38 kinase  (Cell Signaling Technology Inc)

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 periodontal research

Article Title: Pyrophosphate inhibits periodontal ligament stem cell differentiation and mineralization through MAPK signaling pathways

doi: 10.1111/jre.12911

Figure Lengend Snippet: The protein expressions of phosphorylated/unphosphorylated ERK1/2, JNK, and p38 of PDLSCs treated with PPi. (A) Western blot images of phosphorylated/unphosphorylated ERK1/2 and β-actin, with quantification the relative expression of phosphorylated ERK1/2. 15 and 60 min: *p < 0.05 compare to 0 μM group. (B) Western blot images of phosphorylated/unphosphorylated JNK and β-actin, with quantification the relative expression of phosphorylated JNK. 7.5 min: *p < 0.05 compare to 0 μM group; 15 min: *p < 0.05 compare to 100 μM group. (C) Western blot images of phosphorylated/unphosphorylated p38 and β-actin, with quantification the relative expression of phosphorylated p38. *p < 0.05 compare to 0 μM group in each time points; ** p < 0.05 compare to 10 μM group in each time points

Article Snippet: The primary antibodies were as follows: β-actin (1:1000, Santa cruz biotechnology), anti-osteopontin (1:1000, ab8448, Abcam), anti-collagen 1 (1:1000, ab260043, Abcam), anti-DMP1 (1:1000, ab103203, Abcam), anti-osterix (1:1000, ab22552, Abcam), anti-phos-ERK (1:1000, #4376, CST), anti-ERK (1:1000, #9102, CST), anti-phos-Stress-Activated Protein Kinase (SAPK)/JNK (1:1000, #9258, CST), anti-SAPK/JNK(1:1000, #4668, CST), anti-phosphorylated p38 (1:1000, #4511, CST), anti-p38 (1:1000, #8690, CST).

Techniques: Western Blot, Expressing

Journal: Journal of periodontal research

Article Title: Pyrophosphate inhibits periodontal ligament stem cell differentiation and mineralization through MAPK signaling pathways

doi: 10.1111/jre.12911

Figure Lengend Snippet: The protein expressions of osteogenic markers of PDLSC with PPi treatment after inhibiting ERK1/2, JNK, and p38 pathway for 21 days. (A) Western blot images of OPN, RUNX2, OSX, DMP1, and β-actin. (B-E) The relative expressions of OPN, RUNX2, DMP1, and OSX. In (B), *p < 0.05 was compared to ERK 1/2 inhibitor group. In (C), *p < 0.05 was compared to PPi only group. In (D), *p < 0.05 was compared to PPi only group. In (E), *p < 0.05 was compared to PPi only group; **p < 0.05 was compared to the ERK1/2 inhibitor group

Article Snippet: The primary antibodies were as follows: β-actin (1:1000, Santa cruz biotechnology), anti-osteopontin (1:1000, ab8448, Abcam), anti-collagen 1 (1:1000, ab260043, Abcam), anti-DMP1 (1:1000, ab103203, Abcam), anti-osterix (1:1000, ab22552, Abcam), anti-phos-ERK (1:1000, #4376, CST), anti-ERK (1:1000, #9102, CST), anti-phos-Stress-Activated Protein Kinase (SAPK)/JNK (1:1000, #9258, CST), anti-SAPK/JNK(1:1000, #4668, CST), anti-phosphorylated p38 (1:1000, #4511, CST), anti-p38 (1:1000, #8690, CST).

Techniques: Western Blot

Journal: Biomedicines

Article Title: Bag-1 Protects Nucleus Pulposus Cells from Oxidative Stress by Interacting with HSP70

doi: 10.3390/biomedicines11030863

Figure Lengend Snippet: Evaluation of the phosphorylation of MAPK family members and Raf-1 in NP cells. Western blotting ( A ) and quantification ( B , C ) of the phosphorylated forms of p38, ERK, JNK, and Raf-1in NP cells treated with or without H 2 O 2 for 30 min. Data are the means ± SD n = 3, * p < 0.05.

Article Snippet: The membranes were blocked with blocking buffer (5% bovine serum albumin, 0.1% NaN 3 in PBS) and incubated overnight at 4 °C with antibodies against Bag-1 (#ab32109; Abcam plc), p38 (#8690; Cell Signaling Technology), phosphorylated p38 (#4511; Cell Signaling Technology), ERK (#4695; Cell Signaling Technology), phosphorylated ERK (#4370; Cell Signaling Technology), JNK (#9252; Cell Signaling Technology), phosphorylated JNK (#AF1205; R&D Systems, Minneapolis, MN, USA), β-actin (#A2228; Sigma-Aldrich), or GAPDH (#G9545; Sigma-Aldrich).

Techniques: Western Blot

Journal: Biomedicines

Article Title: Bag-1 Protects Nucleus Pulposus Cells from Oxidative Stress by Interacting with HSP70

doi: 10.3390/biomedicines11030863

Figure Lengend Snippet: Evaluation of the phosphorylation of MAPK family members and Raf-1 in Bag-1-overexpressing NP cells. Western blot analysis ( A ) and quantification ( B , C ) of the phosphorylated forms of p38, ERK, JNK, and Raf-1 in NP cells treated with or without H 2 O 2 for 30 min. Data are the means ± SD n = 3, * p < 0.05. ( D ) Comparison of the expression of pERK in NP cells (NP) and Bag-1-overexpressing NP cells (Bag) without H 2 O 2 treatment. Data are the means ± SD n = 3, * p < 0.05.

Article Snippet: The membranes were blocked with blocking buffer (5% bovine serum albumin, 0.1% NaN 3 in PBS) and incubated overnight at 4 °C with antibodies against Bag-1 (#ab32109; Abcam plc), p38 (#8690; Cell Signaling Technology), phosphorylated p38 (#4511; Cell Signaling Technology), ERK (#4695; Cell Signaling Technology), phosphorylated ERK (#4370; Cell Signaling Technology), JNK (#9252; Cell Signaling Technology), phosphorylated JNK (#AF1205; R&D Systems, Minneapolis, MN, USA), β-actin (#A2228; Sigma-Aldrich), or GAPDH (#G9545; Sigma-Aldrich).

Techniques: Western Blot, Expressing

Journal: Biomedicines

Article Title: Bag-1 Protects Nucleus Pulposus Cells from Oxidative Stress by Interacting with HSP70

doi: 10.3390/biomedicines11030863

Figure Lengend Snippet: Evaluation of the phosphorylation of MAPK family members and Raf-1 in thioflavin-S-treated NP cells. Western blot analysis ( A ) and quantification ( B , C ) of the phosphorylated forms of p38, ERK, JNK, and Raf-1 in thioflavin-S-treated NP cells treated with or without H 2 O 2 for 30 min. Data are the means ± SD n = 3, * p < 0.05. ( D ) Comparison of the expression of pERK in NP cells (NP) and thioflavin-S-treated NP cells (ThioS) without H 2 O 2 treatment. Data are the means ± S.D. n = 3, * p < 0.05.

Article Snippet: The membranes were blocked with blocking buffer (5% bovine serum albumin, 0.1% NaN 3 in PBS) and incubated overnight at 4 °C with antibodies against Bag-1 (#ab32109; Abcam plc), p38 (#8690; Cell Signaling Technology), phosphorylated p38 (#4511; Cell Signaling Technology), ERK (#4695; Cell Signaling Technology), phosphorylated ERK (#4370; Cell Signaling Technology), JNK (#9252; Cell Signaling Technology), phosphorylated JNK (#AF1205; R&D Systems, Minneapolis, MN, USA), β-actin (#A2228; Sigma-Aldrich), or GAPDH (#G9545; Sigma-Aldrich).

Techniques: Western Blot, Expressing

Journal: PLoS ONE

Article Title: A20 Is Critical for the Induction of Pam3CSK4-Tolerance in Monocytic THP-1 Cells

doi: 10.1371/journal.pone.0087528

Figure Lengend Snippet: (A) Western blot analysis of A20 expression in scramble siRNA-, or A20 siRNA-transfected THP-1 cells, pre-treated (Pre-T) with medium, or 1 µg/ml Pam3CSK4 (P3C4) for 24 h, and re-treated (Re-T) with 1 µg/ml Pam3CSK4 for 60 min. (B) Real-time RT-PCR analysis of IL-1β, and IL-8 gene expression in scramble siRNA, or A20 siRNA-transfected THP-1 cells, treated as described as (A). * P <0.05 compared with no pre-treatment groups. (C) Western blot analysis of p38, and JNK phosphorylation in scramble siRNA-, or A20 siRNA-transfected THP-1 cells, pre-treated with medium, or 1 µg/ml Pam3CSK4 for 24 h, and re-treated (Re-T) with 1 µg/ml Pam3CSK4 for 30 min. (D) Real-time RT-PCR analysis of A20 gene expression in mock, or A20-expressing plasmid-transfected THP-1 cells. (E) RT-PCR analysis of TNF-α, IL-1β, and IL-8 gene expression in mock, or A20-expressing plasmid-transfected THP-1 cells, treated with medium, or 1 µg/ml Pam3CSK4, or 1 µg/ml LPS for 1 h. (F) Real-time RT-PCR analysis of TNF-α, and IL-8 gene expression in mock, or A20-expressing plasmid-transfected THP-1 cells, treated as described as (E). * P <0.05 compared with the Mock-transfected groups.

Article Snippet: Rabbit anti-human A20, ERK, β-actin, IκB-α, rabbit anti-human phosphorylated p38, ERK, JNK, were purchased from Cell Signaling Technology (Beverly, MA).

Techniques: Western Blot, Expressing, Transfection, Quantitative RT-PCR, Plasmid Preparation, Reverse Transcription Polymerase Chain Reaction

Journal: PLoS ONE

Article Title: A20 Is Critical for the Induction of Pam3CSK4-Tolerance in Monocytic THP-1 Cells

doi: 10.1371/journal.pone.0087528

Figure Lengend Snippet: (A) The effect of various PAMPs on cytokine gene expression. THP-1 cells were treated with medium, PGN (5 µg/ml), Poly (I:C) (5 µg/ml), LPS (1 µg/ml), flagellin (FGN, 0.1 µg/ml), and Pam3CSK4 (1 µg/ml) for 1 h. The gene expression of cytokines was detected by RT-PCR. β-actin gene expression was detected as loading control. (B) The effect of various PAMPs on the cytokine protein expression. THP-1 cells were treated with indicated PAMPs for 24 h. The cytokine proteins in the supernatant were detected by ELISA. * P <0.05 compared with medium group. (C) The effect of various various PAMPs on A20 protein expression. THP-1 cells were treated with indicated PAMPs for 24 h. A20 protein expression was detected by Western blot. β-actin protein expression was detected as loading control. (D) The effect of Pam3CSK4 pre-treatment on LPS-induced cytokine expression. THP-1 cells, pre-treated (Pre-T) with medium, or 1 µg/ml Pam3CSK4 for 24 h, were re-treated (Re-T) with medium, or 1 µg/ml LPS for 1 h. Cytokine gene expression was detected by qRT-PCR. * P <0.05 compared with LPS-treated alone group. (E) The effect of LPS pre-treatment on Pam3CSK4 (P3C4)-induced cytokine expression. THP-1 cells, pre-treated (Pre-T) with the indicated concentrations of LPS for 24 h, were re-treated (Re-T) with medium, or 1 µg/ml Pam3CSK4 for 1 h. Cytokine gene expression was detected by qRT-PCR. # P <0.05 compared with Pam3CSK4-treated alone group. (F) The effect of PGN, Poly(I:C) (IC), LPS, flagellin (FGN) pre-treatment on Pam3CSK4 (P3C4)-induced phosphorylation of p38 and JNK. THP-1 cells, pre-treated with medium, or 10 µg/ml PGN, or 10 µg/ml Poly(I:C)(IC), or 1 µg/ml LPS, or 100 ng/ml flagellin (FGN) for 24 h, were re-treated with medium, or 1 µg/ml Pam3CSK4 for 30 min. The phosphorylation of p38 and JNK was detected by western blot. β-actin protein was detected as loading control.

Article Snippet: Rabbit anti-human A20, ERK, β-actin, IκB-α, rabbit anti-human phosphorylated p38, ERK, JNK, were purchased from Cell Signaling Technology (Beverly, MA).

Techniques: Expressing, Reverse Transcription Polymerase Chain Reaction, Enzyme-linked Immunosorbent Assay, Western Blot, Quantitative RT-PCR

Journal: PLoS ONE

Article Title: A20 Is Critical for the Induction of Pam3CSK4-Tolerance in Monocytic THP-1 Cells

doi: 10.1371/journal.pone.0087528

Figure Lengend Snippet: (A) RT-PCR analysis of A20 gene expression in THP-1 cells, treated with 20 ng/ml IL-1β for the indicated hours. β-actin gene expression was detected as loading controls. (B) Western blot analysis of A20 protein expression in THP-1 cells, treated with 20 ng/ml IL-1β for the indicated hours. β-actin protein was detected as loading controls. (C) Western blot analysis of p38, and JNK phosphorylation in THP-1 cells, pre-treated (Pre-T) with the indicated concentrations of IL-1β for 24 h, and re-treated (Re-T) with medium, or 1 µg/ml Pam3CSK4 for 30 min. β-actin protein was detected as loading controls. (D) RT-PCR analysis of A20 gene expression in THP-1 cells, treated with 20 ng/ml TNF-α for the indicated hours. β-actin gene expression was detected as loading controls. (E) Western blot analysis of A20 protein expression in THP-1 cells, treated with 20 ng/ml TNF-α for the indicated hours. β-actin protein was detected as loading controls. (F) Western blot analysis of p38, and JNK phosphorylation in THP-1 cells, pre-treated with the indicated concentrations of TNF-α for 24 h, and re-treated with medium, or 1 µg/ml Pam3CSK4 for 30 min. β-actin protein was detected as loading controls.

Article Snippet: Rabbit anti-human A20, ERK, β-actin, IκB-α, rabbit anti-human phosphorylated p38, ERK, JNK, were purchased from Cell Signaling Technology (Beverly, MA).

Techniques: Reverse Transcription Polymerase Chain Reaction, Expressing, Western Blot