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: Nuclear proteins were incubated with biotin-labeled oligonucleotide probes containing the binding sequence for MCP-1, CXCL1 or Cx43 (biotin 3′ end DNA labeling kit, Viagene Biotech Inc.).

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: JASPAR and PROMO analysis of the NF-κBp65, p50 or AP-1 binding sites in the promoter region of MCP-1, CXCL1, or Cx43.

Article Snippet: Nuclear proteins were incubated with biotin-labeled oligonucleotide probes containing the binding sequence for MCP-1, CXCL1 or Cx43 (biotin 3′ end DNA labeling kit, Viagene Biotech Inc.).

Techniques: Binding Assay

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: Effect of TAK-242, PDTC or SR11302 on LPS-induced Cx43 expression in cultured astrocytes. (A) Double immunofluorescence indicates the expression of TLR4 on Cx43-labeled dorsal spinal cord astrocytes. Scale bar: 20 μm. (B) RT-qPCR quantitative analysis showed that TAK-242 significantly inhibited LPS-induced Cx43 mRNA expression. * p < 0.05 vs. Veh-treated group, + p < 0.05 vs. Veh + LPS-treated group, n = 5 per group. (C) RT-qPCR quantitative analysis showed that PDTC or SR11302 significantly inhibited LPS-induced Cx43 mRNA expression. * p < 0.05 vs. Veh-treated group, + p < 0.05 vs. Veh + LPS-treated group, n = 5 per group. (D) WB results showed that TAK-242 significantly inhibited LPS-induced Cx43 and p-Cx43 expression. The top panel was the target band, Cx43 and p-Cx43 (Ser368), and the bottom one was for the loading control GAPDH. (E) WB results showed that PDTC or SR11302 significantly inhibited LPS-induced Cx43 and p-Cx43 expression. The top panel was the target band, Cx43 and p-Cx43 (Ser368), and the bottom one was for the loading control GAPDH. (F) Statistical analysis shows the effect of TAK-242 on Cx43 and p-Cx43 protein expression [intensity ratios (normalized to GAPDH expression) relative to the Veh-treated group]. * p < 0.05 vs. Veh-treated group, + p < 0.05 vs. Veh + LPS-treated group, n = 4 per group . (G) Statistical analysis shows the effect of PDTC or SR11302 on Cx43 and p-Cx43 protein expression [intensity ratios (normalized to 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). (H) EMSA analysis was performed on binding reactions (the binding of AP-1 to Cx43 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 (right). * p < 0.05 vs. 4 μg group, + p < 0.05 vs. 8 μg group, n = 4 per group. (I) EMSA analysis was performed on binding reactions (the binding of AP-1 to Cx43 promoter) conducted in LPS-treated cells pretreated with or without PDTC or SR11302. This is a representative of three separate experiments (left). For competition, a 100-fold molar excess of unlabelled 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: Nuclear proteins were incubated with biotin-labeled oligonucleotide probes containing the binding sequence for MCP-1, CXCL1 or Cx43 (biotin 3′ end DNA labeling kit, Viagene Biotech Inc.).

Techniques: Expressing, Cell Culture, Immunofluorescence, Labeling, Quantitative RT-PCR, Standard Deviation, Binding Assay, Activity Assay

Journal: STAR Protocols

Article Title: Protocol to identify the signaling pathways of quorum sensing system in Burkholderia cenocepacia

doi: 10.1016/j.xpro.2024.103381

Figure Lengend Snippet: A graphical depiction illustrating the identification of transcriptional regulatory proteins (A) Express the DsfR protein in E. coli BL21 (DE3). Perform affinity purification of His-fusion proteins using His-Trap affinity columns according to the manufacturer’s instructions. (B) Harvest B. cenocepacia H111 bacterial cells and induce cell lysis to release chromatin. Purify chromatin DNA and sonicate it to yield soluble fragments averaging 200–500 bp in length. Store this chromatin at −20°C as input DNA. Use His-tagged DsfR protein for immunoprecipitation with an anti-His antibody. Generate sequencing libraries from the immunoprecipitated DNA and sequence them to identify motifs. (C) Use biotin as a marker to label the purified promoter DNA fragment at its 3′ end to create a probe sample. Prepare DNA-protein binding reactions by coincubating biotin-labeled probes with proteins. Separate DNA-protein complexes from unbound probes using a 5% polyacrylamide gel. Detect biotin-labeled probes with different mobilities on the membrane. Figure reprinted with permission from Li et al., 2024. Created with Figdraw.

Article Snippet: Biotin 3′ end DNA labeling kit , KeyGEN BioTECH , Cat#KGS132.

Techniques: Affinity Purification, Lysis, Immunoprecipitation, Sequencing, Marker, Purification, Protein Binding, Labeling, Membrane

Journal: STAR Protocols

Article Title: Protocol to identify the signaling pathways of quorum sensing system in Burkholderia cenocepacia

doi: 10.1016/j.xpro.2024.103381

Figure Lengend Snippet:

Article Snippet: Biotin 3′ end DNA labeling kit , KeyGEN BioTECH , Cat#KGS132.

Techniques: Virus, Subcloning, Recombinant, Lysis, Clone Assay, Plasmid Preparation, DNA Extraction, Gel Extraction, Labeling, DNA Labeling, Software, Electroporation, Electrophoresis