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. 2019 Jun 11;9:8462. doi: 10.1038/s41598-019-44934-5

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© The Author(s) 2019

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

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Detection of TARDBP-core promoter interaction in an electrophoretic mobility shift assay (EMSA). (a) Western blot analysis of the recombinant GST-fused TARDBP protein purified using glutathione agarose beads. The protein was detected using antibodies directed against the GST tag and TARDBP protein. (b) The nucleotide sequence of TARDBP binding site within the HBV core promoter region is shown (nt1804-nt1849). The sense (S) and antisense (AS) probes of the sequence, together with the five AS mutant probes (Mutant 1–5), and the S and AS sequence of the adjacent probe (nt1765-nt1810) used for the EMSA assay are indicated. The nucleotide changes in the mutants are marked in red. (c) The AS (WT) probe was incubated alone (lane 1) or with increasing concentrations of TARDBP protein (lanes 2–5); the adjacent AS probe was incubated with 2 μg of TARDBP (lane 6); the AS (WT) probe was incubated with 2 μg of TARDBP alongside increasing concentrations of an unlabeled specific competitor at 0, 1, 2, 10, 50 and 200 fold respectively, (lanes 7–12). (d) A supershift assay was performed with an anti-TARDBP antibody; lane 1, AS (WT) probe alone; lane 2, AS (WT) probe with TARDBP protein; lane 3 AS (WT) probe with TARDBP protein along with 2 μg rabbit anti-TARDBP antibody. The specific TARDBP-DNA complex (shifted probe arrow), and the antibody-TARDBP-DNA complex (super shifted probe arrow) is indicated. (e) The HBV-producing T23 cells were transfected with 5 μg of FLAG tagged TARDBP, immunoprecipated with an anti-FLAG antibody and blotted with anti-FLAG and anti-TARDBP antibodies. The arrow shows the location of the exogenous protein, and the asterisk (*) shows the endogenous protein which was also precipitated by the FLAG-antibody. (f) Starting at the N-terminus, mutations were introduced at five sites in the AS (WT) probe to make five different mutant probes, Mutants 1–5, as shown in (b). The WT probe was incubated alone (lane 1) or in the presence of 2 μg of TARDBP protein (lane 2), while each of the five mutant probes was also incubated with the equivalent amount of TARDBP protein (lanes 3–7). The location of the free probe and the specific TARDBP-DNA complex (shifted probe) are indicated by arrows.