Figure 5

Physical interaction between POSH and Siah2. a: The AH-109 yeast strain was co-transformed with full length POSH Gal4-DNA-binding-domain fusion construct (pGBKT7-POSH) and wild type Siah2 GAL4-activation-domain fusion construct (pACT-Siah2) or empty vector controls. Transformants were grown on plates supplemented with (+Ade+His) or without (-Ade-His) adenine and histidine. -Ade-His is the yeast-medium-plate lacking Leu, Trp, His and Ade in which activation of the adenine and histidine reporter gene is being tested. +Ade+His is the yeast-medium-plate lacking Leu and Trp, selecting only for presence of plasmids being tested for interaction. b: PPC-1 cells stably expressing POSH-myc were transfected with rmSiah2-HA or caspase-8 p30 as a negative control and co-immunoprecipitation using anti-myc conjugated magnetic beads was performed. The anti-myc immune complexes were analyzed for the presence of Siah2-myc via Western blot. c: Schematic representation of full length POSH (top) and various mutants cloned into the pGBKT7 vector. The yeast strain AH-109 was co-transformed with pACT-Siah2 and the various POSH mutants and grown on +Ade+His or -Ade-His plates. YES/NO indicates the presence or absence of colonies on -Ade-His plates (left).