Figure 2
From: PIAS1 interacts with FLASH and enhances its co-activation of c-Myb
PIAS1 functions as a SUMO E3 ligase and co-activator of FLASH. (A) CV-1 cells were transfected with plasmids encoding 3×FLAG-FLASH-D or 3×FLAG-FLASH-D-KR (0.5 μg), (His)6-SUMO-1 (0.5 μg) and increasing amounts (0.5, 1.0, 1.5 μg) of HA-PIAS1 (left panel). Likewise, CV-1 cells were transfected with the same plasmids, except for (His)6-SUMO-1 which was exchanged with 0.5 μg GFP-SUMO-1 (right panel). Whole cell extracts were analyzed by SDS-PAGE and immunoblotting using anti-FLAG antibody for the detection of FLASH, as well as anti-PIAS1 and anti-GAPDH antibodies. Arrows indicate non-sumoylated FLASH (F), sumoylated FLASH (S-F), and FLASH sumoylated with GFP-SUMO1 (GS-F). (B) CV-1 cells were transfected with plasmids encoding Gal4p-DBD-FLASH wild-type, K1813R mutant (FLASH-KR), or Gal4p-DBD only (0.4 μg) in absence and presence of PIAS1 wild-type or PIAS1 RING finger mutant (PIAS1-C350S) (0.2 μg) in combination with a Gal4p-driven SNRPN promoter reporter construct. The results are presented as relative luciferase units (RLU) and represent the mean RLU ± SEM of three independent assays performed in triplicates. (C) GST, GST-FLASH-D wild-type (GST-FLASH) and K1813R (GST-FLASH KR) were incubated with lysates from COS-1 cells transfected with full-length 3×FLAG-tagged PIAS1 wild-type or RING finger mutant (C346S/C351S/H353A/C356S). PIAS1 was detected by an anti-FLAG antibody. 5% of the input (total cell extract) used for the pulldown was loaded as reference. The amount of GST and GST fusion proteins was evaluated with Ponceau S red staining of the membrane after immunoblotting. */**, GST or GST-FLASH-D, respectively.