Journal: The Journal of biological chemistry

Article Title: The Fatty Acid-binding Protein, aP2, Coordinates Macrophage Cholesterol Trafficking and Inflammatory Activity

doi: 10.1074/jbc.M413788200

Figure Lengend Snippet: A, kinase activity of IKK is impaired by aP2 deficiency. The activity of IKK was determined by in vitro kinase phosphorylation of an IκBα-GST substrate after macrophage lines were stimulated by 1 μg/ml LPS for the indicated times. Western blot analysis of immunoprecipitated IKKα/β is shown on the right. B, reconstitution of aP2 restores kinase activity of IKK. aP2+/+, aP2−/−, and aP2−/−R macrophage cell lines were stimulated by 1 μg/ml LPS for 10 min, and kinase activity of IKK was assayed as in A. Data shown are representative of three independent kinase assays. C, NF-κB binding to DNA is abrogated by aP2 deficiency. EMSA was used to determine NF-κB DNA binding activity of nuclear protein extracts from aP2+/+ and aP2−/− macrophages (−) stimulated with 1 μg/ml LPS (+) for 30 min using radiolabeled or non-labeled (cold competition, CC) NF-κB consensus-binding sequences (top panel). Nuclear extracts from the Burkitt's lymphoma cell line, Raji, served as a positive control. Western blot analysis of NF-κB in untreated macrophage lines (bottom panel). D, NF-κB driven reporter activity is impaired by aP2 deficiency. Macrophage lines were co-transfected with pNF-κB-Luc and phRL-null-Luc reporter constructs and activated with 1 μg/ml LPS (top panel) or with 0 (unstim), 25, 50, or 75 μg of membrane extracts from CHO-control cells or CHO-CD154 (bottom panel) for 16 h. Relative promoter activities are reported as the ratio of firefly to Renilla luciferase activity ± S.D. of triplicate samples (top) or fold induction by CHO-CD154 relative to CHO control (bottom).

Article Snippet: DNA binding activity of NF-κB was determined using the Gelshift Plus Rel Family electrophoretic mobility shift assay (EMSA) kit (Geneka Biotechnology, Inc., Montreal, Canada). aP2 +/+ and aP2 −/− macrophages lines were stimulated with 1 μg/ml LPS for 30 min. Nuclear proteins were extracted using a modified Dignam method ( 26 ).

Techniques: Activity Assay, In Vitro, Phospho-proteomics, Western Blot, Immunoprecipitation, Binding Assay, Labeling, Positive Control, Transfection, Construct, Membrane, Control, Luciferase

Journal: Frontiers in Genetics

Article Title: A T > G Mutation in the NR5A2 Gene Is Associated With Litter Size in Hu Sheep Through Upregulation of Promoter Activity by Transcription Factor MTF-1

doi: 10.3389/fgene.2019.01011

Figure Lengend Snippet: Competitive EMSA assay confirms the specific binding between MTF-1 and -700GG of the NR5A2 promoter region. Probes in this assay containing the NR5A2 -700GG site with or without biotin labeling were called bio-probes or cold probes, and probes containing the NR5A2 -700TT site without biotin labeling were called mut-probes. These probes were incubated with ovary nuclear extracts in different combinations, and DNA–protein complexes were visualized by autoradiography. FP, free probes; NSB, non-specific binding; DNA–protein complex: DNA and transcription factor MTF-1 binding complex.

Article Snippet: EMSAs were performed according to the instructions provided in the Competitive EMSA Kit (Viagene Biotech, Changzhou, China).

Techniques: Binding Assay, Labeling, Incubation, Autoradiography

Journal: Virology Journal

Article Title: Identification of virion-associated transcriptional transactivator (VATT) of SGIV ICP46 promoter and their binding site on promoter

doi: 10.1186/s12985-019-1210-0

Figure Lengend Snippet: Electrophoretic mobility shift assay. The whole protein extractions from AraC-pretreated and SGIV-infected GS cells (AraC+SGIV, lane 3), CHX-pretreated and SGIV-infected GS cells (CHX + SGIV, lane 4), SGIV-infected GS cells (SGIV+GS, lane 5), GS cells (GS, lane 6) and purified SGIV virion (SGIV, lane 7) were obtained for EMSA analysis with biotin-labeled probe X that contains the sequence of SGIV ICP46 promoter. Incubated with biotin-labeled probe NFκB, the nuclear extracts with activated NFκB was used as positive control (lane 1) and nuclear extracts without activated NFκB (lane 2) was used as negative control. Arrow show the DNA binding proteins; NSB, non-specific binding; P, free biotin labeled probe

Article Snippet: After incubated with the protein extraction from purified SGIV virion, the EMSA were performed using Non-Radioactive EMSA Kits (Viagene Biotech, Changzhou, China) following the manufacturer’s protocol.

Techniques: Electrophoretic Mobility Shift Assay, Infection, Purification, Labeling, Sequencing, Incubation, Positive Control, Negative Control, DNA Binding Assay, Binding Assay

Journal: Virology Journal

Article Title: Identification of virion-associated transcriptional transactivator (VATT) of SGIV ICP46 promoter and their binding site on promoter

doi: 10.1186/s12985-019-1210-0

Figure Lengend Snippet: Determination of VATT binding site on SGIV ICP46 promoter by EMSA. Probe X that contains the 64 bp SGIV ICP46 promoter were divided into five probes which were overlapped each other continuously (probe 1–5 in Table ). After incubated with the protein extraction from purified SGIV virion, the EMSA were performed. Arrow show the DNA binding proteins of SGIV. Incubated with biotin-labeled probe NFκB, the nuclear extracts with activated NFκB was used as positive control (lane 9) and nuclear extracts without activated NFκB (lane 10) was used as negative control

Article Snippet: After incubated with the protein extraction from purified SGIV virion, the EMSA were performed using Non-Radioactive EMSA Kits (Viagene Biotech, Changzhou, China) following the manufacturer’s protocol.

Techniques: Binding Assay, Incubation, Protein Extraction, Purification, DNA Binding Assay, Labeling, Positive Control, Negative Control