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. 2015 Jul 8;290(34):21019–21031. doi: 10.1074/jbc.M115.639476

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© 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

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FIGURE 5. — RCAN1 modulates PACAP-induced neuronal differentiation. A, PC12 cells were transfected with an empty (pcDNA), RCAN1 knockdown (shRCAN1#1), or RCAN1 overexpression (RCAN1.4) vector, and transfected cells were selected either in G418- or puromycin-containing growth medium. Stable clones were identified by immunoblotting using an anti-RCAN1 antibody. B, phase-contrast images of PC12 cells treated with PACAP (10 nm) on days 0 and 4. Scale bars = 30 μm. C, differentiation was evaluated by calculating the percentage of neurite-bearing cells versus the total number of cells in a field. Cells with at least one neurite with a length greater than the diameter of the cell body were counted as neurite-bearing cells. The results are the mean ± S.D. of three independent experiments. D, PC12 cells were transfected with the indicated shRNA expression vectors, and the protein extracts were immunoblotted with anti-RCAN1 antibody. E, differentiation was evaluated as in C. ***, p < 0.001; n.s., not significant. F and G, stable cells were treated with PACAP (10 nm) for the indicated times, and the cell extracts were immunoblotted with anti-phospho ERK, anti-ERK, anti-Synapsin-1, and GAPDH antibodies. H, genotyping results of a WT and a Ts65Dn trisomy mouse. M, marker. I and J, the cortical neurons of a wild-type and a TS65Dn mouse were treated with PACAP (10 nm), and the cell extracts were immunoblotted with the indicated antibodies. K, schematic summarizing the participation of RCAN1.4 in PACAP-mediated neuronal differentiation.

FIGURE 5. — RCAN1 modulates PACAP-induced neuronal differentiation. A, PC12 cells were transfected with an empty (pcDNA), RCAN1 knockdown (shRCAN1#1), or RCAN1 overexpression (RCAN1.4) vector, and transfected cells were selected either in G418- or puromycin-containing growth medium. Stable clones were identified by immunoblotting using an anti-RCAN1 antibody. B, phase-contrast images of PC12 cells treated with PACAP (10 nm) on days 0 and 4. Scale bars = 30 μm. C, differentiation was evaluated by calculating the percentage of neurite-bearing cells versus the total number of cells in a field. Cells with at least one neurite with a length greater than the diameter of the cell body were counted as neurite-bearing cells. The results are the mean ± S.D. of three independent experiments. D, PC12 cells were transfected with the indicated shRNA expression vectors, and the protein extracts were immunoblotted with anti-RCAN1 antibody. E, differentiation was evaluated as in C. ***, p < 0.001; n.s., not significant. F and G, stable cells were treated with PACAP (10 nm) for the indicated times, and the cell extracts were immunoblotted with anti-phospho ERK, anti-ERK, anti-Synapsin-1, and GAPDH antibodies. H, genotyping results of a WT and a Ts65Dn trisomy mouse. M, marker. I and J, the cortical neurons of a wild-type and a TS65Dn mouse were treated with PACAP (10 nm), and the cell extracts were immunoblotted with the indicated antibodies. K, schematic summarizing the participation of RCAN1.4 in PACAP-mediated neuronal differentiation.