Figure 7.

THRB2^H10 specifically affects negative regulation of gene expression. (a) Expression levels of WT THRB2 and THRB2^H10 in Thrb KD TαT1.1 cells were tested by western blotting with an anti-HA antibody. Relative expression of (b and c) negatively and (d and e) positively regulated genes were measured by qRT-PCR and normalized to Actb mRNA (see Materials and Methods) and compared with controls (scrambled KD, GFP, and Thrb KD GFP, Fig. 2). Reexpression of HA-THRB2 in Thrb KD cells reconstituted (b) Tshb, and (c) Rxrg negative regulation, with the maximal percentage of repression (left panes) 75% to 80%. Expression of HA-THRB2^H10 only partially rescued negative regulation of Tshb and Rxrg (45% to 50% of repression at 10 nM T3). WT THRB and THRB2^H10 showed similar properties in activation of gene expression of (d) Rab 27 and (e) Sema3c. No significant differences (NS) were seen in percentage of activation (right panels) at 1 and 10 nM T3. Left panels show relative gene expression [y-axis (log₂)] with increased concentration of T3 [x-axis (log10); y intersects x at 0 nM). Expression levels were normalized to those in scramled KD, GFP no T3. Data points are presented as mean ± standard error of the mean. Differences were considered to be significant at P < 0.05.