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. Author manuscript; available in PMC: 2011 Jul 20.
Published in final edited form as: Dev Cell. 2010 Jul 20;19(1):138–147. doi: 10.1016/j.devcel.2010.06.008

Figure 3.

Figure 3

RhoGEF19 is a direct target gene of GRHL3. (A) EMSA of recombinant (r) GRHL3 binding to the RhoGEF19 promoter. A 100-fold molar excess of unlabelled cold competitor probe was added in the indicated lanes. The migration of the specific GRHL3/DNA complexes is arrowed. (B) ChIP analysis of GRHL3 on the RhoGEF19 promoter. Chromatin from HEK 293T cells transfected with empty vector (lane 5) or HA-tagged Grhl3 (lane 6) was immunoprecipitated using antisera to the HA-tag. As negative controls, we used pre-immune sera (lanes 3 and 4). Lane 1 and 2 shows the input chromatin. The immunoprecipitated chromatin was amplified with RhoGEF19, or control MyoD primers. (C) Q-PCR of RhoGEF19 expression in wild type (hatched boxes) and Grhl3−/− (closed boxes) E18.5 epidermis and dermis. Bars represent standard errors. The HPRT levels served as a reference. (D, E) GRHL3 induced RhoA activation in HEK 293T cells is mediated through increased RhoGEF19 expression. HA-tagged Grhl3 was transfected into HEK 293T cells transduced with an shRNA to RhoGEF19, or a Scrambled control shRNA (Scr), and expression of HA-GRHL3 was confirmed by Western blot (IB:α-HA, α-actin loading control). GTP-Rho was precipitated using GST-RBD and detected by anti-RhoA antibody (IB:αRhoA). Total RhoA was also detected in lysates with anti-RhoA antibody. HA-GRHL3 is arrowed. *non-specific band with HA antibody. Expression of human RhoGEF19 in Scr control cells, Scr cells expressing HA-GRHL3, and RhoGEF19-kd cells expressing HA-GRHL3 was determined by Q-PCR. See also Figure S3.