Fig. 4.
Phosphorylation of Thr-210 of TR2 triggers its association with PML in P19. (A) P19 cells were transiently transfected (0.5 μg/ml, 16 h) with flag-tagged WT TR2, flag-tagged negative SUMOylation mutation in combination with phosphomimetic (210CP + K238R) or negative phosphorylation mutation (210CN + K238R). Association with PML was examined by coimmunoprecipitation experiment (panel 1). Transfection input was monitored in panel 2, and actin is shown in panel 3. (B) The association of endogenous TR2 with PML in P19 was examined in the presence of atRA (0.1 μM, 12 h) and/or reagents triggering ERK2 activation (ERK2-Ac) or inhibition ERK2-In. (C) A kinetic experiment monitoring endogenous TR2 association with PML (IP followed by IB) in P19 treated with atRA. Corresponding changes in Oct4 gene expression were shown in the bottom panel. Total PML expression was also monitored. (D) Immunostaining of endogenous TR2 and PML in control cells, or cells treated with atRA (0.1 μM) for 6 h. Nuclei were stained with DAPI. Large colocalized TR2/PML puncta in stimulated cells are marked with arrows. Shown at the bottom is the statistical result by scoring the percentage of cells with colocalized TR2 and PML puncta (positive) among the total cells (positive + negative).