The APOE-mediated translation response is regulated by calcium signature and its sources. A, Rat primary cortical neurons (DIV15) were treated with MK801 (25 μm) along with APOE3 or APOE4 (10–15 nm) conditioned media for 1 min and probed for phosphorylation of eEF2. Left, Representative immunoblots indicating levels of phospho-eEF2, eEF2, and Tuj1. Right, Graph indicating ratio of phospho-eEF2 to eEF2 normalized to Tuj1. Data are represented as mean ± SEM, N = 5, one-way ANOVA (p = 0.0006) followed by Dunnett's multiple comparison test. B, Rat primary cortical neurons (DIV15) were treated with MK801 (25 μm) along with APOE KO conditioned media for 1 min and probed for the phosphorylation of eEF2. Left, Representative immunoblots indicating levels of phospho-eEF2, eEF2, and Tuj1. Right, Graph indicating ratio of phospho-eEF2 to eEF2 normalized to Tuj1. Data are represented as mean ± SEM, N = 5. Unpaired Student's t test (ns - non-significant). C, Rat primary cortical neurons (DIV15) were treated with nifedipine (50 μm) along with APOE3 or APOE4 (10–15 nm) conditioned media for 1 min and probed for phosphorylation of eEF2. Left, Representative immunoblots indicating levels of phospho-eEF2, eEF2, and Tuj1. Right, Graph indicating ratio of phospho-eEF2 to eEF2 normalized to Tuj1. Data are represented as mean ± SEM, N = 3, one-way ANOVA (p = 0.001) followed by Dunnett's multiple comparison test. D, Rat primary cortical neurons (DIV15) were treated with nifedipine (50 μm) along with APOE KO conditioned media for 20 min and probed for phosphorylation of eEF2. Left, Representative immunoblots indicating levels of phospho-eEF2, eEF2, and Tuj1. Right, Graph indicating ratio of phospho-eEF2 to eEF2 normalized to Tuj1. Data are represented as mean ± SEM, N = 4. Unpaired Student's t test (ns - non-significant). E, Rat primary cortical neurons (DIV15) were treated with MK801 (25 μm) along with recombinant APOE3 protein (15 nm) for 1 min. They were subjected to FUNCAT along with immunostaining for MAP2. The representative images for MAP2 and FUNCAT fluorescent signal under different treatment conditions are shown (scale bar: 10 μm). F, The graph represents the quantification of the FUNCAT fluorescent intensity normalized to MAP2 fluorescent intensity under different APOE3 treatment conditions. Each data point represents an individual neuron. N = 40–50 neurons from 4 independent experiments, one-way ANOVA (p < 0.0001) followed by Tukey's multiple comparison test. G, Rat primary cortical neurons (DIV15) were treated with MK801 (25 μm) along with recombinant APOE4 protein (15 nm) for 1 min. They were subjected to FUNCAT along with immunostaining for MAP2. The representative images for MAP2 and FUNCAT fluorescent signal under different treatment conditions are shown (scale bar: 10 μm). H, The graph represents the quantification of the FUNCAT fluorescent intensity normalized to MAP2 fluorescent intensity under different APOE4 treatment conditions. Each data point represents an individual neuron. N = 40–50 neurons from 4 independent experiments, one-way ANOVA (p < 0.0001) followed by Tukey's multiple comparison test. I, Rat primary cortical neurons (DIV15) were treated with nifedipine (50 μm) along with APOE3 or APOE4 (10–15 nm) conditioned media for 20 min and probed for phosphorylation of eEF2. Top, Representative immunoblots indicating levels of phospho-eEF2, eEF2, and Tuj1. Bottom, Graph indicating ratio of phospho-eEF2 to eEF2 normalized to Tuj1. Data are represented as mean ± SEM, N = 3, one-way ANOVA (p = 0.0036) followed by Tukey's multiple comparison test. J, Rat primary cortical neurons (DIV15) were treated with nifedipine (50 μm) along with recombinant APOE3 or APOE4 protein (15 nm) for 20 min. They were subjected to FUNCAT along with immunostaining for MAP2. The representative images for MAP2 and FUNCAT fluorescent signal under different treatment conditions are shown (scale bar: 10 μm). K, The graph represents the quantification of the FUNCAT fluorescent intensity normalized to MAP2 fluorescent intensity under different treatment conditions. Each data point represents an individual neuron. N = 20–30 neurons from 4 independent experiments, one-way ANOVA (p < 0.0001) followed by Tukey's multiple comparison test.