nf κbp50  (Santa Cruz Biotechnology)

Journal: PLoS ONE

Article Title: Effects of Soluble Epoxide Hydrolase Deficiency on Acute Pancreatitis in Mice

doi: 10.1371/journal.pone.0113019

Figure Lengend Snippet: A ) Total pancreas lysates from wild type mice without (n = 6) and with (n = 9) cerulein, and Ephx2 KO mice without (n = 6) and with (n = 9) cerulein were immunoblotted for pIKKα, pIκBα, pNF-κB and their respective unphosphorylated proteins, NF-κBp50 and Tubulin as a loading control. Representative immunoblots (n = 2–3 samples per group) are shown. Bar graphs represent normalized data for pIKKα/IKKα, pIκBα/IκBα, pNF-κB/NF-κB and NF-κBp50/Tubulin as means±SEM (AU: arbitrary units). (*: P≤0.05; **: P≤0.01) indicate significant difference between mice without and with cerulein administration, and (##: P≤0.01) indicates significant difference between WT and KO mice. B ) Total pancreas lysates from wild type mice without (n = 8) and with (n = 8) arginine administration for the indicated times, and Ephx2 KO mice without (n = 8) and with (n = 8) arginine administration were immunoblotted for pIKKα, pIκBα, pNF-κB and their respective unphosphorylated proteins, NF-κBp50 and Tubulin. Representative immunoblots (n = 2–3 samples per group) are shown. Bar graphs represent normalized data for pIKKα/IKKα, pIκBα/IκBα, pNF-κB/NF-κB and NF-κBp50/Tubulin as means±SEM (AU: arbitrary units). (**: P≤0.01) indicates significant difference between mice without and with arginine administration, and (#: P≤0.05; ##: P≤0.01) indicates significant difference between WT and KO mice.

Article Snippet: Immunoblotting of lysates and immunoprecipitates was performed with antibodies for sEH (generated by the Hammock laboratory), cleaved Caspases 8, 9 and 3, PARP, SHP1 and Tubulin (all from Santa Cruz), pp38 (Thr180/Tyr182), p38, pJNK (Thr183/Tyr185), JNK, pIKKα/β (Ser178/180), IKKα/β, pIκBα (Ser32), IκBα, pNF-κBp65 (Ser536), NF-κBp65 and NF-κBp50 (all from Cell Signaling).

Techniques: Western Blot

Journal: Frontiers in Molecular Neuroscience

Article Title: NF-κB and AP-1 are required for the lipopolysaccharide-induced expression of MCP-1, CXCL1, and Cx43 in cultured rat dorsal spinal cord astrocytes

doi: 10.3389/fnmol.2022.859558

Figure Lengend Snippet: List of antibodies and their information used in this study.

Article Snippet: Astrocytes cultures were stained with rabbit anti-NF-κBp65 antibody (520 μg/ml, 1:350, Proteintech Group, Rosemont, IL, United States), mouse anti-NF-κBp50 antibody (200 μg/ml, 1:350, Santa Cruz, Dallas, TX, United States) or rabbit anti-AP-1 antibody (1200 μg/ml, 1:350, Proteintech Group, Rosemont, IL, United States) overnight.

Techniques:

Journal: Frontiers in Molecular Neuroscience

Article Title: NF-κB and AP-1 are required for the lipopolysaccharide-induced expression of MCP-1, CXCL1, and Cx43 in cultured rat dorsal spinal cord astrocytes

doi: 10.3389/fnmol.2022.859558

Figure Lengend Snippet: TAK-242 suppresses LPS-induced nuclear translocation of NF-κBp50 and the expression of NF-κBp50, IκB-α, and IKKα in cultured astrocytes. Cells were pretreated with TAK-242 (100 nM, 2 h) and then stimulated with LPS (1 μg/ml, 24 h). (A) Immunofluorescence staining images showed NF-κBp50 localization in astrocytes. Scale bar: 50 μm. (B) The percentage of nuclear p50-positive cells among the total cells was quantified. * p < 0.05 vs. Veh-treated group, + p < 0.05 vs. Veh + LPS-treated group, n = 10 fields (at least 50 cells were counted per condition). (C) WB image of NF-κBp50, IκB-α, and IKKα protein expression. The top panel was the target band, NF-κBp50, IκB-α, and IKKα, and the bottom one was for the loading control β-actin/GAPDH. (D) Statistical analysis of the protein expression of NF-κBp50, IκB-α, and IKKα [intensity ratios (normalized to β-actin or GAPDH expression) relative to the Veh-treated group]. * p < 0.05 vs. Veh-treated group, + p < 0.05 vs. Veh + LPS-treated group, all data were expressed as mean ± standard deviation of at least four independent experiments ( n = 4 per group).

Article Snippet: Astrocytes cultures were stained with rabbit anti-NF-κBp65 antibody (520 μg/ml, 1:350, Proteintech Group, Rosemont, IL, United States), mouse anti-NF-κBp50 antibody (200 μg/ml, 1:350, Santa Cruz, Dallas, TX, United States) or rabbit anti-AP-1 antibody (1200 μg/ml, 1:350, Proteintech Group, Rosemont, IL, United States) overnight.

Techniques: Translocation Assay, Expressing, Cell Culture, Immunofluorescence, Staining, Control, Standard Deviation

Journal: Frontiers in Molecular Neuroscience

Article Title: NF-κB and AP-1 are required for the lipopolysaccharide-induced expression of MCP-1, CXCL1, and Cx43 in cultured rat dorsal spinal cord astrocytes

doi: 10.3389/fnmol.2022.859558

Figure Lengend Snippet: The treatment with PDTC or SR11302 decreased LPS-induced NF-κBp65, NF-κBp50, and AP-1 DNA binding activity in cultured astrocytes. (A) EMSA analysis was performed on binding reactions (the binding of NF-κBp65 to MCP-1 promoter) conducted in the presence of the indicated μg protein amount of Veh + LPS-treated cells (left). NP, no protein; UP, unlabeled probe; NS, non-specific band. NF-κBp65 DNA binding activity in Veh + LPS-treated cells was analyzed (right). * p < 0.05 vs. 4 μg group, + p < 0.05 vs. 8 μg group, n = 4 per group. (B) EMSA analysis was performed on binding reactions (the binding of NF-κBp65 to MCP-1 promoter) conducted in LPS-treated cells pretreated with or without PDTC or SR11302. For competition, a 100-fold molar excess of unlabelled probe (comp) was added to nuclear extracts from Veh + LPS group. This is a representative of three separate experiments (left). NF-κBp65 DNA binding activity in different groups was analyzed (right). * p < 0.05 vs. Veh-treated group, + p < 0.05 vs. Veh + LPS-treated group, n = 4 per group. (C) EMSA analysis was performed on binding reactions (the binding of NF-κBp50 to MCP-1 promoter) conducted in the presence of the indicated μg protein amount of Veh + LPS-treated cells (left). NP, no protein; UP, unlabeled probe. NF-κBp50 DNA binding activity was analyzed. * p < 0.05 vs. 4 μg group, + p < 0.05 vs. 8 μg group, n = 4 per group. (D) EMSA analysis was performed on binding reactions (the binding of NF-κBp50 to MCP-1 promoter) conducted in LPS-treated cells pretreated with or without PDTC or SR11302. For competition, a 100-fold molar excess of unlabeled probe (comp) was added to nuclear extracts from Veh + LPS group. This is a representative of three separate experiments (left). NF-κBp50 DNA binding activity in different groups was analyzed (right). * p < 0.05 vs. Veh-treated group, + p < 0.05 vs. Veh + LPS-treated group, n = 4 per group. (E) EMSA analysis was performed on binding reactions (the binding of AP-1 to CXCL1 promoter) conducted in the presence of the indicated μg protein amount of Veh + LPS-treated cells (left). NP, no protein; UP, unlabeled probe. AP-1 DNA binding activity was analyzed. * p < 0.05 vs. 4 μg group, + p < 0.05 vs. 8 μg group, n = 4 per group. (F) EMSA analysis was performed on binding reactions (the binding of AP-1 to CXCL1 promoter) conducted in LPS-treated cells pretreated with or without PDTC or SR11302. For competition, a 100-fold molar excess of unlabeled probe (comp) was added to nuclear extracts from Veh + LPS group. AP-1 DNA binding activity in different groups was analyzed (right). * p < 0.05 vs. Veh-treated group, + p < 0.05 vs. Veh + LPS-treated group, n = 4 per group.

Article Snippet: Astrocytes cultures were stained with rabbit anti-NF-κBp65 antibody (520 μg/ml, 1:350, Proteintech Group, Rosemont, IL, United States), mouse anti-NF-κBp50 antibody (200 μg/ml, 1:350, Santa Cruz, Dallas, TX, United States) or rabbit anti-AP-1 antibody (1200 μg/ml, 1:350, Proteintech Group, Rosemont, IL, United States) overnight.

Techniques: Binding Assay, Activity Assay, Cell Culture

Journal: Frontiers in Molecular Neuroscience

Article Title: NF-κB and AP-1 are required for the lipopolysaccharide-induced expression of MCP-1, CXCL1, and Cx43 in cultured rat dorsal spinal cord astrocytes

doi: 10.3389/fnmol.2022.859558

Figure Lengend Snippet: The treatment with PDTC or SR11302 decreased LPS-induced NF-κBp65 activity in cultured astrocytes. Cells were pretreated with PDTC (1 μM, 30 min) or SR11302 (10 μM, 2 h) and then stimulated with LPS (1 μg/ml, 24 h). (A) Immunofluorescence staining images showed NF-κBp65 localization. Scale bar: 50 μm. (B) The percentage of nuclear NF-κBp65-positive cells among the total cells was quantified. * p < 0.05 vs. Veh-treated group, + p < 0.05 vs. Veh + LPS-treated group, n = 10 fields (at least 50 cells were counted per condition). (C) RT-qPCR quantitative analysis of NF-κBp65 mRNA expression. * p < 0.05 vs. Veh-treated group, + p < 0.05 vs. Veh + LPS-treated group, n = 5 per group. (D) RT-qPCR quantitative analysis of NF-κBp50 mRNA expression. * p < 0.05 vs. Veh-treated group, + p < 0.05 vs. Veh + LPS-treated group. (E) WB images of NF-κBp65, p-NF-κBp65, NF-κBp50, IκB-α, and IKKα protein expression. The top panel was the target band, NF-κBp65, p-NF-κBp65, NF-κBp50, IκB-α and IKKα, and the bottom one was for the loading control β-actin/GAPDH. (F) Statistical analysis of the protein expression of NF-κBp65, p-NF-κBp65, NF-κBp50, IκB-α, and IKKα [intensity ratios (normalized to β-actin or GAPDH expression) relative to the Veh-treated group]. * p < 0.05 vs. Veh-treated group, + p < 0.05 vs. Veh + LPS-treated group, all data were expressed as mean ± standard deviation of at least four independent experiments ( n = 4 per group).

Article Snippet: Astrocytes cultures were stained with rabbit anti-NF-κBp65 antibody (520 μg/ml, 1:350, Proteintech Group, Rosemont, IL, United States), mouse anti-NF-κBp50 antibody (200 μg/ml, 1:350, Santa Cruz, Dallas, TX, United States) or rabbit anti-AP-1 antibody (1200 μg/ml, 1:350, Proteintech Group, Rosemont, IL, United States) overnight.

Techniques: Activity Assay, Cell Culture, Immunofluorescence, Staining, Quantitative RT-PCR, Expressing, Control, Standard Deviation