Figure 4.

USP36 SUMOylates Las1L. A, Las1L is SUMOylated in cells. H1299 cells transfected with Flag-Las1L in the absence or presence of His-SUMO1 or His-SUMO2 were subjected to Ni²⁺-NTA PD under denaturing conditions, followed by IB with anti-Flag, anti-SUMO1, and anti-SUMO2/3 antibodies. B, USP36 promotes Las1L SUMOylation. H1299 cells transfected with the indicated plasmids were subjected to Ni²⁺-NTA PD, followed by IB to detect Las1L SUMOylation. The SUMOylated Las1L and total SUMOylated proteins are indicated. The protein expression is shown in the bottom panels. C, Knockdown of endogenous USP36 reduces Las1L SUMOylation. H1299 cells transfected with the indicated plasmids together with scr or USP36 siRNA were subjected to Ni²⁺-NTA PD, followed by IB to detect Las1L SUMOylation. The protein expression is shown in the bottom panels. D and E, Las1L is SUMOylated at K565. H1299 cells transfected with WT Las1L or the indicated lysine mutant plasmids were subjected to Ni²⁺-NTA beads PD under denaturing conditions, followed by IB. The SUMOylated Las1L and total SUMOylated proteins are indicated. The protein expression is shown in the bottom panels. The relative ratio of SUMOylated Las1L to native Las1L normalized to WT Las1L from the four individual experiments are shown in E.