Supplemental Data

Generation of PIKE knockout mice (PIKE -/-) In order to study the neuronal functions of PIKE-L on GluA2/GRIP1 association in vivo, we generated mice with targeted disruption in the CENTG1 locus via homologous recombination using LoxP/Cre system. We first made a transgenic line with the loxP insertions in intron 2 and 7 of CENTG1 gene (Fig S4A). PIKE flox/+ mice were then bred with transgenic mice that express Cre recombinase in all tissues. Deletion of exons 3 to 6 resulted in removal of GTPase domain as well as introducing a frame shift mutation that created a new stop codon, leading to a truncated PIKE protein for all isoforms. Heterozygous mating generated newborn pups at expected Mendelian frequency that appeared indistinguishable from the wild-type littermates. Moreover, PIKE-deficient mice were viable, fertile, and of normal size and weight at birth (data not shown). We confirmed the ablation of PIKE-L protein by RT-PCR (Fig S4B) and Western blot analysis using anti-PIKE-L antibody (Fig S4C). Immunohistochemical staining using an antibody recognizing the C-terminus of PIKE-L revealed that no signal was detected in PIKE-null cortex, which further confirmed the successful removal of PIKE in the knockout lines (Fig S4D). PI3K activity was substantially reduced in brain extract from PIKE -/mice (Fig S4E, top panel). In agreement with the reduced PI3K activity, Akt phosphorylation was also diminished (Fig S4E, middle panel).