Figure 1
From: PIAS1 interacts with FLASH and enhances its co-activation of c-Myb
FLASH and PIAS1 interact and co-localize in nuclear speckles. (A) AH109 yeast cells were transformed with bait vector pDBT or pDBT-FLASH-D and mated with the yeast strain Y187 pretransformed with prey vector pGADT7 or pGADT7-PIAS1[1-501]. Interaction was verified by activation of the HIS3 reporter gene (SC-L-W-H). Each mating was performed in duplicate. (B) AH109 yeast cells were transformed with bait vector pDBT or pDBT-FLASH-A/B/C/D/E and mated with Y187 pretransformed with prey vector pGADT7 or pGADT7-PIAS1. Interaction was verified by activation of the HIS3 reporter gene (SC-L-W-H). Each mating was performed in duplicate. (C) Schematic overview of the regions of FLASH used in yeast two-hybrid assays. Abbreviation; DRD = DED-recruiting domain [3]. A: amino acids 1-304, B: 305-867, C: 868-1507, D: 1508-1982, E: 1814-1982. (D) GST, GST-FLASH-D and GST-FLASH-A were incubated with lysate from COS-1 cells transfected with full-length 3×FLAG-tagged PIAS1. PIAS1 was detected by an anti-FLAG antibody. 5% of the input (total cell extract) used for the pulldown was loaded as reference. (E) Lysates from COS-1 cells transfected with GFP-FLASH and 3×FLAG-PIAS1 or empty vector, were immunoprecipitated with anti-FLAG antibody or an irrelevant antibody (anti-GST), and the precipitates were analyzed by immunoblotting using anti-GFP (FLASH) and anti-FLAG (PIAS1) for detection. 5% (PIAS1) or 10% (FLASH) of the input used for immunoprecipitation was loaded as reference. ProtG, protein G-Sepharose beads only. (F) HEK 293 cells were transfected with plasmids expressing 3×FLAG-PIAS1 and a Gal4p-DBD-FLASH fusion protein. Occupancies of PIAS1 on the 5×GRE promoter and on the NCOA5 intron were analysed using ChIP-qPCR. (G) CV-1 cells were transfected with plasmids encoding HA-PIAS1 and 3×FLAG-FLASH, and analysed with indirect immunofluorescence and confocal microscopy. PIAS1 was detected with rabbit anti-HA antibody and Alexa Fluor 488 goat anti-rabbit IgG. FLASH was detected with mouse anti-FLAG antibody and Alexa Fluor 633 goat anti-mouse IgG1. The merged image is shown in the right panel.