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. 2020 Oct 20;48(20):11452–11467. doi: 10.1093/nar/gkaa905

Figure 5.

Figure 5.

Phosphorylation of Msx1 at Ser136 is essential for its binding to and upregulation of Fgf9 and Fgf18 and further activation the MAPK signaling pathway, and CDK1 appears to be the kinase that performs this phosphorylation. (A) Western blotting to assess the effect of Msx1 phosphorylation sites on increases in the levels of p-Erk1/2, Fgf9 and Fgf18 by wild-type Msx1. (B) qRT-PCR assays to assess the effect of Msx1 phosphorylation sites on increases in the levels of Fgf9 and Fgf18 by wild-type Msx1. Values are the means ± SD. ***P < 0.0001. (C) ChIP-qPCR analysis to determine the impact of Ser136 mutants of Msx1 on Fgf9/18 binding. ChIP assays were performed with C2C12 cells overexpressing wild-type Msx1, Msx1 (S136A), Msx1 (S136D) or the control. ChIP-qPCR was then used to determine the relative enrichment of the Msx1 binding fragments of the Fgf9 and Fgf18 genes. The ChIP-qPCR data were analyzed to calculate the enrichment of the control or different Msx1 mutants relative to input respectively, and the control values were defined as 1 to calculate the fold enrichment of Msx1 or its mutants over the control. Values are the means ± SD. ***P < 0.0001, **P < 0.001, *P < 0.01. (D) Diagram of the conserved residues of the CDK1 catalytic domain and the corresponding phosphorylated peptide sequence of Msx1. (E) Western blotting to examine the effect of Msx1 or its mutants and Ro3306 on the level of p-Erk1/2 in C2C12 cells. C2C12 cells overexpressing wild-type Msx1, Msx1(S136A), Msx1(S136D) or the control were treated with 10 μM Ro3306 or DMSO. Twenty hours later, the cells were harvested and lysed for western blotting, and the level of p-Erk1/2 was checked. (F) Western blotting to verify the specificity of Ser136 phosphorylated Msx1 (p-Msx1) antibody. Lysates from C2C12 cells overexpressing Flag-tagged Msx1 were incubated with calf intestinal alkaline phosphatase (CIAP) at 37°C for half an hour and subjected to immunoblot analysis with the indicated antibodies. (G) Western blotting to examine the effect of Ro3306 on the level of endogenous p-Msx1 in C2C12 cells. C2C12 cells were treated with 10 μM Ro3306 or DMSO. Twenty hours later, the cells were harvested and lysed for western blotting with the indicated antibodies.