Figure 6.

Smad4 enhances the Taz interaction with Runx2 or PPARγ. (A, F): HA‐Runx2 or Flag‐PPARγ plasmid was cotransfected with the Flag‐Taz or HA‐Taz plasmid into HEK293 cells in the absence or presence of Flag‐Smad4, according to the indicated combinations. (B, G): Flag‐Smad4 plasmid was cotransfected with the HA‐Runx2 (B) or HA‐PPARγ (G) into HEK293 cells in the absence or presence of Flag‐Taz. (C, H): After osteogenic differentiation for 3 days (C) or adipogenic differentiation for 4 days (H) of Smad4‐knockdown and shGFP‐expressing C3H10T1/2 cells, cell lysates were IP with anti‐Taz antibody and immunoblotted. (D, I): pcDNA‐Runx2 (D) or pcDNA‐PPARγ (I) plasmid was cotransfected with the mOC‐Luc or aP2‐Luc reporter, together with Flag‐Taz, into HEK293 cells under dose‐dependent expression of Flag‐Smad4. Expressions of the indicated proteins were confirmed by IB. Luciferase activity was normalized to the expression of Renilla luciferase. (E, J): After osteogenic (E) or adipogenic (J) differentiation of Smad4‐knockdown and control C3H10T1/2 cells, chromatin immunoprecipitation assays were performed on day 3 (E) or day 4 (J). Chromatin fragments were IP with anti‐Taz antibody. Polymerase chain reaction primers for the osteocalcin promoter region or aP2 promoter were used to amplify the DNAs. The amplified DNAs from input samples were used for normalizing the data. IgG: negative control. The data were analyzed by one‐way analysis of variance followed by Tukey's multiple comparison test (*p < .05, **p < .01 and ***p < .001 compared to the indicated controls, n = 3). Bars represent the mean ± SD. Images in this figure are representative of three independent experiments. Abbreviations: AM, adipogenic differentiation medium; IB, immunoblot; IgG, immunoglobulin G; IP, immunoprecipitated; OM, osteogenic differentiation medium; PPARγ, peroxisome proliferator‐activated receptor‐γ; Runx2, runt‐related transcription factor 2; shGFP, green fluorescent protein‐specific short hairpin RNA; TCL, total cell lysates.