Figure 4: SUMOylation stabilizes TARBP2 by reducing ubiquitination.

(a) SUMOylation of TARBP2 reduces its ubiquitination. 293T cells transfected with indicated plasmids were lysed with RIPA buffer. Cell lysates were used for IP with anti-myc antibody and followed by western blot analysis with anti-HA antibody to detect ubiquitination of TARBP2. Lysates as an Input were also immunoblotted with anti-myc, anti-Flag, anti-HA and anti-GAPDH antibodies. (b) SUMOylation of TARBP2 represses its ubiquitination. 293T cells were transfected with HA-Ubiquitin and Flag-TARBP2-WT or -K52R. IP with anti-Flag antibody and western blot analysis were performed as before. (c) SUMOylation of TARBP2 mainly inhibits K48-linked ubiquitination. 293T cells transfected with Flag-TARBP2 and HA-Ubiquitin were lysed and used for IP with anti-Flag antibody. K48R indicated HA-Ubiquitin that bore a mutation on Lysine48 to Arginine. (d) SUMOylation of TARBP2 increases its stability. HeLa and 293T cell lines stably expressing Flag-TARBP2-WT or -K52R were seeded at the same cell density and cultured for 24 h before the treatment with CHX (100 μg ml⁻¹) with indicated time course. Cell lysates were analysed using western blot analysis with anti-Flag and anti-GAPDH antibodies. The line charts presented for the relative fold of TARBP2 were analysed using ImageJ. (e) The SUMOylation level is positively corrected with the expression of TARBP2. Stable Senp1 or SUMO1 knock down 293T cell lines were generated and the expression levels of endogenous TARBP2 in these cell lines were analysed using western blot analysis with anti-TARBP2 antibody. (f) TARBP2 has a longer half-life under higher SUMOylation level. Stable Senp1 knock down and control 293T cell lines were treated with CHX (100 μg ml⁻¹) with indicated time course. Cell lysates were analysed using western blot analysis. The line charts presented for the relative fold of TARBP2 are analysed using ImageJ. For full scans of western blots (a,b,d), see Supplementary Fig. 8.