Fig. 3.

RanBP2 promotes multiple conjugation of both SUMO1 and SUMO2/3 on TRIM5α. a Schematic representation of the experimental procedure. b HeLa-SUMO1, and c HeLa-SUMO2/3 cells were transduced with shBP2, shE, or not transduced (NT). After 3 days, RanBP2 depletion was assessed by qPCR. TRIM5α was immunoprecipitated from d HeLa-SUMO1 and e HeLa-SUMO2/3 cells. Input samples were analyzed by immunoblotting with a α-His antibody to reveal SUMO-modified proteins, while TRIM5α immunoprecipitates were probed with α-SUMO1 or α-SUMO2/3 antibodies. Higher molecular weight bands indicate SUMO-modified TRIM5α (TRIM5α*S1 and TRIM5α*S2/3). IgG heavy (HC) and light chains (LC) are indicated. Western blots are representative of 3 independent experiments (original blots in Supplementary Figure 4). f TRIM5α*S1 bands were quantified from 10 independent SUMO blots of TRIM5α immunoprecipitates. The graph shows the mean relative intensity of SUMO bands normalized for NT controls ± SEM. Statistical analysis was performed using a paired t test