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. 2016 Mar 25;7(18):26220–26234. doi: 10.18632/oncotarget.8354

Figure 4. Effect of sumoylation on the half-life of TCF21, subcellular localization and its interaction with ERα and HDAC1/2.

Figure 4

A. MCF-7 cells transfected with Flag-tagged wild-type or mutant TCF21(K24R) were treated with 10 mg/ml CHX at the indicated time periods. Cell lysate was subjected to Western blot assay. The graph shows the relative intensity of the TCF21 band at different time points. The level of TCF21 protein in control cells was set to 1. Data shown in the graphs are the means ± SDs of three independent experiments. ‘*’ indicates significantly different from cells transfected with mutant TCF21 (K24R) at the P<0.05 level. B. HEK 293T cells transfected with Myc-Ub and Flag-tagged wild-type TCF21 or mutant TCF21(K24R) were collected, and then subjected to IP using anti-Flag antibody followed by Western blot with anti-Myc or anti-Flag antibody. C. MCF-7 cells transfected with Flag-tagged wild-type or mutant TCF21(K24R) were stained with rabbit anti-Flag antibody (red) and then counterstained with DAPI (blue) for nucleus detection. D. MCF-7 cells were transfected with Flag-tagged wild-type or mutant TCF21(K24R). Cytosolic and nuclear fractions were subjected to Western blot using anti-Flag antibody. Fibrillarin and β-actin were measured to monitor the efficiency of nuclear and cytosolic preparations, respectively. E. HEK 293T cells transfected with EGFP-ERα and Flag-tagged wild-type or mutant TCF21(K24R) were collected, and then subjected to IP using anti-Flag antibody followed by Western blot with anti-GFP or anti-Flag antibody. F. HEK 293T cells transfected with Myc-HDAC1 and Flag-tagged wild-type or mutant TCF21(K24R) were collected, and then subjected to IP using anti-Flag antibody followed by Western blot with anti-Myc or anti-Flag antibody. G. HEK 293T cells transfected with Myc-HDAC2 and Flag-tagged wild-type or mutant TCF21 (K24R) were collected, and then subjected to IP with anti-Flag antibody followed by Western blot with the anti-Myc or anti-Flag antibody. H. MCF-7 cells transfected with Flag-tagged wild-type or mutant TCF21(K24R) were subjected to IP using anti-ERα antibody and re-IP with anti-Flag antibody followed by Western blot with anti-HDAC1, anti-HDAC2, anti-Flag or anti-ERα antibody.