SiRNA knockdown of dynein IC2-C recovered the inhibition of neurite outgrowth in NF1-KD PC12 cells. siRNA knockdown of dynein IC2-C recovered the inhibition.

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Supplementary File S3 A EGFP PC12 cells SOCS6 PC12 cells

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siRNA knockdown of dynein IC2-C recovered the inhibition of neurite outgrowth in NF1-KD PC12 cells. siRNA knockdown of dynein IC2-C recovered the inhibition of neurite outgrowth in NF1-KD PC12 cells. PC12 cells were transfected for 24 h with control siRNA, NF1 (249) siRNA, both NF1 (249) and dynein IC2-C (329) siRNAs, or both NF1 (249) and dynein IC2-C (331) siRNAs before treatment with NGF. After 48-h NGF treatment, dynein expression in the cells was analyzed by 2-D immunoblotting using anti-dynein IC antibody, and the neurite length of the cells was measured. A, Representative spot patterns of dynein IC2 in 2D immunoblotting of PC12 cells treated with control siRNA [a], NF1 (249) siRNA [b], NF1 (249) siRNA, and dynein IC2-C (329) siRNA [c], and NF1 (249) siRNA and dynein IC2-C (331) siRNA [d]. B, Differential interference contrast images of PC12 cells treated with control siRNA [a], NF1 (249) siRNA [b], NF1 (249) siRNA and dynein IC2-C (329) siRNA [c], and NF1 (249) siRNA and dynein IC2-C (331) siRNA [d]. C, Measurement of neurite length of PC12 cells treated with the siRNAs. The average of the total length of PC12 neurites are shown on the y axis. Data are expressed as means and S.E. of three independent experiments. For each experiment, more than 50 cells were counted. D, Comparison of the intensity of each dynein IC2 spot after transfection with NF1 siRNA and dynein IC2-C (329) siRNA. The histogram shows the percentage ratio of the intensity of each spot to the total spot intensity of dynein IC2 in individual samples. The intensity of each sample was obtained by 2D-immunoblotting using anti-dynein IC antibody followed by Cy5-labeled secondary antibody. The data were obtained from the average values of three separate identical experiments. Error bars represent S.E. of each set of three experiments. Mio Hirayama et al. Mol Cell Proteomics 2013;12:1377-1394 © 2013 by The American Society for Biochemistry and Molecular Biology, Inc.