Fig. 3. The C-terminal domain of Miranda is required for its ubiquitylation. (A) Constructs encoding FLAG-Miranda or FLAG-Miranda in which the C-terminal 103 amino acids were deleted (FLAG-Miranda^Cterm) under the control of an actin promoter were cotransfected into Drosophila S2 cells with DNA encoding HA-ubiquitin under the control of a heat-shock promoter. Extracts were prepared from cells both without (-) and with (+) heat shock. FLAG-Miranda but not FLAG-Miranda^Cterm was detected in the immune complex after the cells were heat shocked. (B) FLAG-Miranda and FLAG-Miranda^Cterm were expressed in S2 cells, and cell extracts were subjected to immunoprecipitation (IP) with either anti-FLAG or anti-ßgal antibodies followed by detection of ubiquitylated protein by western blotting using anti-ubiquitin antibody. Ubiquitylated Miranda was detected in the immune complex after immunoprecipitation of FLAG-Miranda but not FLAG-Miranda^Cterm. (C) FLAG-tagged Miranda was expressed in larval neuroblasts (NBs), and larval brain extracts were immunoprecipitated with either anti-FLAG or anti-ßgal antibodies; ubiquitylated protein was detected using anti-ubiquitin antibody.