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: Cholesterol-25-hydroxylase (CH25H) expression following treatment of astrocytes with thrombin. (A) Primary astrocytes stained with an antibody against glial fibrillary acid protein (GFAP; Alexa Fluor 488-labeled donkey anti-mouse IgG, green) and Hoechst 33342 showing a purity of over 95%. Scale bar: 100 μm. (B) Western blot analysis of CH25H expression following astrocyte stimulation with 1 U/mL thrombin for 24, 48, or 72 hours. Quantities were normalized to β-actin. (C) 3-(4,5)-Dimethylthiahiazo(-z-y1)-3,5-di-phenytetrazoliumromide (MTT) assay of astrocyte viability after stimulation with 0–50 μM argatroban for 24 hours. (D) Western blot analysis of CH25H expression following astrocyte stimulation with 1 U/mL thrombin in the presence or absence of 0–20 μM Argatroban for 24 hours. Quantities were normalized to β-actin. (E) Quantitative polymerase chain reaction analysis of par1 , par3 , and par4 expression in primary astrocytes. Quantities were normalized to gapdh . (F) MTT assay of astrocyte viability after stimulation with 0–5 μM SCH79797 for 24 hours. (G) Western blot analysis of CH25H following astrocyte stimulation with 1 U/mL thrombin in the presence or absence of 0–3 μM SCH79797 for 24 hours. Quantities were 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). PAR: Protease-activated receptor; SCH79797: PAR1 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, Staining, Labeling, Western Blot, MTT Assay, Real-time Polymerase Chain Reaction

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