Fig 2: Creation of recombinant, GST-tagged Fmrp and Pum2 and validation of APC/C and mTOR association with PUM1 A, B Representative western blot of PUM1 pulled down by recombinant (A) GST-FMRP or (B) GST-PUM2.CRepresentative western blot of Rptor, Cad, Anapc1 and Fzr1 proteins pulled down by IP against Pum1 (used here as positive control) from WT mice.DRepresentative western blot of the reciprocal co-IPs against Anapc1, Fzr1, Rptor, and Cad. Each co-IP was immunoblotted against Pum1 and the pulled down protein used here as a reference protein to confirm the respective protein–protein interaction. Data information: For (C) and (D), IP against IgG was used as a negative control, and Input (1% from the initial protein lysate) as a loading control. Molecular weights are expressed in kilodaltons (kDa) to the right. All mice were sacrificed at 10 weeks of age with an equal number or male and female.

Fig 3: Many Pum1 interactors are specific to certain brain regions and not determined by expression level AProteomic analysis of the whole brain, cerebellum, hippocampus, and cortex at 10 weeks of age shows that Pum1 interactors are not the most highly expressed proteins. Dotted line represents the mean expression of Pum1 interactors from each brain region. Proteomics was performed in duplicate (one male and one female) for each brain region.BAbsolute expression value of the validated Pum1 interactors. Pum2 is expressed at low levels in all three brain regions but was still the strongest Pum1 interactor in the cortex, suggesting a specific interaction rather than a post-lysis artifact.CImmunoblot for Pum1 (positive control), Anapc1, Cnot1 and Pum2.DWestern blot analysis at 10 weeks of age to evaluate Pum2 expression levels in eight different brain regions as well as whole brain. Pum2 is highly expressed in the olfactory bulbs and amygdala, and expressed at similar levels in the hippocampus, cerebellum, and cortex. Data information: Cerebellar and cortical tissues: n = 8 wild-type mice (four male and four female), for a total of 24 mice. Hippocampus: n = 10 wild-type mice (five female and five male), for a total of 30 mice. All mice were 10 weeks of age. IP against IgG was used as a negative control. Molecular protein weights are expressed in kilodaltons (kDa). AM, amygdala; BS, brain stem; CB, cerebellum; CX, cortex; HP, hippocampus; MB, midbrain; OB, olfactory bulbs; SN, substantia nigra pars compacta; WB, whole brain. All the experiments were repeated at least three times.