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. 2020 Aug 3;11(9):2630–2638. doi: 10.1111/1759-7714.13593

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© 2020 The Authors. Thoracic Cancer published by China Lung Oncology Group and John Wiley & Sons Australia, Ltd.

This is an open access article under the terms of the http://creativecommons.org/licenses/by/4.0/ License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited.

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Transcriptional regulation of mAb NJ001 on the TIMP‐3 gene in SPC‐A1 cells. (a) The DNA sequences of TIMP‐3 promoter region were cloned into luciferase reporter constructs: pTIMP‐3 (⁻702/+18), pTIMP‐3 (mutP53), pTIMP‐3 (mutFOXP1) and pTIMP‐3 (mutE2F). (b) TIMP‐3 promoter activity was analyzed with a luciferase reporter assay in SPC‐A1 cells. Data are expressed as the means ± s.d. Control, NJ001; *P < 0.001, compared with pTIMP‐3 (⁻702/+18) or luciferase reporter constructs not treated with NJ001. (c) Electrophoretic mobility shift assay (EMSA) to analyze the activity of nuclear proteins at FOXP1 binding sites in the TIMP‐3 promoter region. Biotinylated probes (20 fmol) were incubated with 8 mg of nuclear extracts from SPC‐A1 cells. In competition experiments, 10‐, 100‐, and 200‐fold molar excess of unlabeled A/B probes show the specificity of each binding reaction.

Inline graphic — Transcriptional regulation of mAb NJ001 on the TIMP‐3 gene in SPC‐A1 cells. (a) The DNA sequences of TIMP‐3 promoter region were cloned into luciferase reporter constructs: pTIMP‐3 (⁻702/+18), pTIMP‐3 (mutP53), pTIMP‐3 (mutFOXP1) and pTIMP‐3 (mutE2F). (b) TIMP‐3 promoter activity was analyzed with a luciferase reporter assay in SPC‐A1 cells. Data are expressed as the means ± s.d. Control, NJ001; *P < 0.001, compared with pTIMP‐3 (⁻702/+18) or luciferase reporter constructs not treated with NJ001. (c) Electrophoretic mobility shift assay (EMSA) to analyze the activity of nuclear proteins at FOXP1 binding sites in the TIMP‐3 promoter region. Biotinylated probes (20 fmol) were incubated with 8 mg of nuclear extracts from SPC‐A1 cells. In competition experiments, 10‐, 100‐, and 200‐fold molar excess of unlabeled A/B probes show the specificity of each binding reaction.