(A) Gallery of HA-immunofluorescence images showing the respective subcellular localization of HSS-tagged GID subunits MAEA, Twa1 and WDR26, stably expressed from a doxycycline-inducible promoter. Co-staining of DNA with DAPI visualizes the nuclei. The proteins are predominantly nuclear with some diffuse staining in the cytoplasm. Scale bar, 5 μM. (B) Immunoprecipitation of HEK-293 control and MAEA-KO cells transiently expressing HSS-tagged Rmnd5a. The presence of associated proteins was examined by immunoblotting with the indicated antibodies. The Twa1 antibody also recognizes an unspecific band at approximately the same size in WCEs (asterisks). Note that the assembly of the GID complex requires the RING protein MAEA. (C) Cell extracts prepared from RPE control (ctrl), MAEA-KO (targeted by two distinct sets of gRNAs, left panel) or WDR26-KO cells (right panel) were analyzed 6 days after lentiviral treatment for the indicated cell cycle and growth pathway markers by immunoblotting with the indicated antibodies. Note that cells deficient for GID activity show reduced levels of phosphorylated (780 and 807/811) and total levels of the tumor suppressor protein Rb, Cyclin A and phosphorylated Histone H3 indicating that they stopped proliferating. The autoregulated novel GID subunit YPEL5 is stabilized in catalytically inactive MAEA-KO cells. (D) Depletion efficiency of individual GID subunits in mouse primary hepatocytes analyzed by qRT-PCR using gene-specific primers. Averaged data plotted from independent experiments ± SEM, n = 2. (E) Relative mRNA expression of FBP1 in murine primary hepatocytes in gluconeogenic and glycolytic conditions in control siRNA or GID siRNA samples analyzed by qRT-PCR. Graphs present the averaged data from two independent experiments performed in duplicates ± SEM, n = 2. (F) The ability of hepatic glucose production was measured in control- and GID siRNA-treated cells starved for 6 hr before processing. Data expressed as mean mg/dl of glucose per μg protein relative to control ± SD, n = 6. (G) Glucose levels in the medium of control and GID-depleted primary hepatocytes were determined 4 hr after they were shifted from gluconeogenesis to glycolysis. Data presented as mean fold change relative to control ± SD, n = 3.