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. 2019 May 30;44(2):491–502. doi: 10.3892/ijmm.2019.4215

Figure 4.

Figure 4

Rap1 downregulates the TGF-β1-induced activation of RhoA. Cells were incubated with 10 ng/ml TGF-β1 for various durations (0, 0.5, 1 and 3 h). (A) GTP-Rap1 was selectively precipitated from cell lysates using the His-RalGDS-RBD fusion protein and Ni-NTA His-Bind resin, and detected by immu-noblotting with an anti-Rap1 antibody. (B) Quantification of the protein expression of GTP-Rap1 (mean ± SEM, n=3, **P<0.01 and ***P<0.001, one-way ANOVA, Tukey's post hoc test). Mock vector, pcDNA-Rap1 N17 (encoding the dominant negative form of Rap1) and pcDNA-Rap1 V12 (encoding the consti-tutively active form of Rap1) were transfected into HSC-T6 cells, and the cells were stimulated with or without 10 ng/ml TGF-β1 for 1 h. GTP-RhoA levels were determined using a pull-down assay with GST-Rhotekin-RBD fusion protein and Glutathione-Sepharose beads. (C) Expression of RhoA was determined by western blotting using an anti-RhoA antibody and (D) quantified (mean ± SEM, n=3, *P<0.05, **P<0.01, and ***P<0.001, one-way ANOVA, Tukey's post hoc test). TGF-β1, transforming growth factor-β1; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; M, mock vector.