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. 2020 Oct 23;3:612. doi: 10.1038/s42003-020-01348-8

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© The Author(s) 2020

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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Fig. 4 — a qPCR of MEF2C mRNA in the subcutaneous fat tissue of MSTN-KO and WT pigs (n = 3 biologically independent animals). b Western blotting of MEF2C protein in the subcutaneous fat tissue of MSTN-KO and WT pigs (n = 3 biologically independent animals). M is the Page Ruler Prestained Protein Ladder 26616 (ThermoFisher Scientific). c qPCR of miRNA222 abundance in response to the ectopic expression of MEF2C. n = 3 independent experiments. “***” Indicates statistically significant. d Western blotting of ectopic MEF2C expression. Flag, flag-tagged MEF2C. M is the Page Ruler Prestained Protein Ladder 26616 (ThermoFisher Scientific). e qPCR of miRNA222 abundance in response to MEF2C silencing. Data shown as the mean ± SD (n = 3 independent experiments). “***” Indicates statistically significant. f Western blotting of MEF2C silencing. M is the Page Ruler Prestained Protein Ladder 26616 (ThermoFisher Scientific). g Schematic of the MEF2C-binding sites on miR222 promoter region. “TATAAATACTT”, native binding motif. “GCCGCTGCACC”, introduced mutant binding motif. h Dual luciferase reporter testing of MEF2C upon miR222 promoter. pGL3-miR222-WT and pGL3-miR222-Mut represent wild-type and mutant version of miR222 promoter, respectively. n = 3 independent experiments. “***” Indicates statistically significant. i Dual luciferase reporter testing of MEF2C silencing upon miR222 transcription. n = 3 independent experiments. “***” Indicates statistically significant. j ChIP-PCR for the in vivo regulation of MEF2C on miR222 transcription. Sites A and B are the predicted MEF2C-binding sites in the miR222 promoter region. Anti-RNA, positive control antibody against RNA polymerase. Anti-MEF2C, antibody against MEF2C. IgG, negative control antibody. Input, sonicated DNA-protein mixture prior to immuno precipitation. PCR Neg. Ctrl and PCR Pos. Ctrl represent the negative and positive controls to demonstrate the primer specificity. M is 100 bp DNA ladder. k Sanger sequencing results for the PCR products from the lane “anti-MEF2C” of site A. l ChIP-qPCR validation of MEF2C binding at miR222 promoter. Anti-RNA and IgG are positive and negative controls, respectively. m MEF2C binding to the miR222 promoter verified in vitro by EMSA. The sequence and chemical modifications are shown at the bottom. WT probe (×150) was used as a competitor.