Figure S3.

Subcellular Fractionation of Primary Rat Astrocytes Reveals Foxo3a Nuclear Translocation

a-d, Immunofluorescence micrographs of rat spinal cord tissue stained for Foxo3a (green) and DNA (DAPI, blue) 3 days after dorsal column (DC) crush and treatment with PBS (DC + vehicle), TFP (DC + CaM_i) or H89 (DC + PKA_i) injected into the lesion site; zoomed-in images are shown for each panel; e, Relative protein kinase activity in cultured primary rat astrocytes subjected to PKA activation with forskolin (10⁻⁵ M) or 10 minutes extracellular hypotonicity (85 mOsm) in the absence or presence of TFP. Data (n = 3) are normalized to untreated controls. ^∗ represents p < 0.05, ns represents p > 0.05 compared to untreated control by ANOVA followed by t test with Bonferroni correction, see Table S2 for p values); f, Immunoblot of fractionated primary rat astrocytes showing the abundance of Foxo3a, which is higher in the nuclear fraction (only degradation products are seen the cytoplasmic fraction as shown in panel g) following PKA activation with forskolin or hypotonicity. This is consistent with the image in panel b showing localization of Foxo3a to nuclei in injured spinal cord tissue. C = cytoplasmic fraction, N = nuclear fraction; g, Primary rat astrocytes were subjected to subcellular fractionation following activation of PKA by forskolin or hypotonicity. Intact Foxo3a was detected only in the nucleus (predicted molecular weight 71 kDa, black arrowhead). Degraded Foxo3a was detected in the cytoplasm (white arrowhead). h, Densitometry of nuclear Foxo3a signals shown in panel g and normalized to nuclear Lamin B. ^∗ represents p < 0.05, ns represents p > 0.05 (see Table S2 for p values). Related to Figure 2.