Subcellular Localization of RAI1 Mutants

To study the expression of the mutant protein, Neuro-2a cells were transfected with Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA) and the plasmid pALTER-MAX RAI1-HA wild type or RAI1-HA c.3440G > A. All transfections were performed according to the manufacturer’s protocol. Western blot analysis was performed as previously described [26 (link)]. Immunodetection was performed using rat anti-HA (1:7000, Roche, Branford, CT, USA) and rabbit anti-β-tubulin (1:1000, sc-9104 Santa Cruz, Dallas, TX, USA). Results were visualized by chemiluminescence. For immunofluorescence, cells were fixed 24 h after transfection with 4% paraformaldehyde followed by permeabilization with 0.2% Triton X-100 in PBS. Subcellular localization of RAI1-HA wild type and mutant forms were detected using the anti-HA high affinity antibody (1:1000, clone 3F10, Roche, Branford, CT, USA). Secondary antibody conjugated to Alexa Fluor 488 (1:1000) was used. Cells were stained with 4′, 6-diamidino-2-phenylindole, dihydrochloride (DAPI) and mounted with a Dako fluorescent mounting medium.