Figure 6.

Set-β interacts with axon growth-suppressing PP2A-A. A, Acutely purified E18 hippocampal neurons' nuclear (N) and cytoplasmic (C) fractions confirm successful separation when immunoblotted for GAPDH (cytoplasmic marker) and histone H3 (nuclear marker; BH, brain homogenate). B, P4 RGCs immunostained at 1 d for endogenous PP2A-A (red) and Set-β (green). Nucleus outlined with dashed white line. Side panel shows Z plane with blue and orange lines indicating x-y plane shown in the middle, demonstrating PP2A-A localization at the membrane and neurites. Scale bar, 20 μm. C, Proteins from E18 hippocampal homogenates coimmunoprecipitated using anti-Set-β antibody or IgG control as marked, immunoblotted for PP2A-A or Set-β as marked. Set-β and PP2A-A immunoprecipitated with anti-Set-β antibody but not with IgG control. D, P4 RGCs transfected with mCherry or DsRed-PP2A-A, were immunostained at 3 d for Tuj1 (neurite marker, green), transfection reporter (red), MAP2 (dendrite marker, data not shown), and counterstained with DAPI (nuclear marker, blue). Nucleus outlined with dashed white line. PP2A-A localized predominantly to the cytoplasm and cellular membranes. Scale bars: 100 μm; insets, 10 μm.