The Journal of Neuroscience, January 3, 2007, ():
DISC1 Regulates the Transport of the NUDEL/LIS1/14-3-3
Complex through Kinesin-1
J. Neurosci. Taya et al.
27: 15
Supplemental Data
Files in this Data Supplement:
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Supplemental Figure 1. Interaction of DISC1 with KIF5s and NUDEL
A, Lysates from transfected COS7 cells expressing HA-KIF5C and Myc-DISC1 were used in an immunoprecipitation assay with antibody against HA. Bound proteins and inputs of assay were analyzed by immunoblotting with antibodies against Myc and HA. B, Lysates from transfected COS7 cells expressing Myc-DISC1, NUDEL-GFP, and GST-14-3-3ε were used in a pull-down assay with GST-14-3-3ε. The expression levels Myc-DISC1, NUDEL-GFP, or GST-14-3-3ε were almost equal in each lane (data not shown). Bound proteins were analyzed by immunoblotting with antibodies against Myc, GFP, and GST. The lower bands represent degradation products of GST-14-3-3ε in immunoblotting with antibody against GST. C, Lysates from transfected COS7 cells expressing EGFP or DISC1-EGFP and Myc-KIF5A-Full, -HL, -C1, -C2 were used in an immunoprecipitation assay with antibody against GFP. Bound proteins and inputs of assay were analyzed by immunoblotting with antibodies against Myc. D, Lysates from transfected COS7 cells expressing EGFP-DISC1 mutants were used in a pull-down assay with GST-KIF5A. Bound proteins and inputs of assay were analyzed by immunoblotting with antibody against GFP.
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Supplemental Figure 2. Inhibition of axon elongation by knockdown of DISC1
A, B, Hippocampal neurons transfected with expression plasmid encoding EGFP-GST and scramble (A) or rDISC1-siRNAs (B) at DIV1 are detected by EGFP fluorescence and the staining of DISC1 (red) and Tau-1 (blue) at DIV3. C, D, Hippocampal neurons transfected with Cy3-labeled scramble (C) or rDISC1-siRNAs (D) are detected by Cy3 fluorescence and staining of DISC1 (green) and Tau-1 (blue). Arrows indicate EGFP-GST or Cy3-labeled siRNA transfected neurons. Bars represent 20 μm.
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Supplemental Figure 3. Effect of DISC1 knockdown on the complex formation of NUDEL/LIS1/14-3-3ε and localization of EB1 in the distal part of the axon
A, PC12 cells were transfected with scramble or rDISC1-siRNAs and then differentiated with NGF for 48 h. Lysates from transfected PC12 cells were used in an immunoprecipitation assay with antibody against NUDEL. Bound proteins and total cell lysates were analyzed by immunoblotting with antibodies against DISC1, NUDEL, LIS1, and 14-3-3ε. Aliquots of original samples (10% Input) and eluates (30%) were subjected to SDS-PAGE. B, Hippocampal neurons transfected with Cy3-scramble and expression plasmid encoding EB1-EGFP at DIV1 are shown by the fluorescence of Cy3 and EGFP and the staining of F-actin (blue) at DIV3. In Cy3-scramble transfected neurons, EB1-EGFP was accumulated in the axonal tips. C, Hippocampal neurons transfected with rDISC1-siRNAs and expression plasmid encoding EB1-EGFP are shown by the fluorescence of EGFP and the staining of DISC1 (red) and F-actin (blue). The enlarged images of the axons are shown.
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Supplemental Figure 4. Effect of DISC1 knockdown on localization of Sra-1 or Numb in the distal part of the axon
A, C, Hippocampal neurons transfected with scramble and expression plasmid encoding EGFP-GST at DIV1 are shown by the fluorescence of EGFP and the staining of F-actin (blue) and Sra-1 (A) or Numb (C) at DIV3. B, D, Hippocampal neurons transfected with rDISC1-siRNAs and expression plasmid encoding EGFP-GST are shown by the fluorescence of EGFP and the staining of F-actin (blue) and Sra-1 (B) or Numb (D). Arrows indicate the distal parts of axons. Bars in the merged panels represent 20 μm. E, F, DISC1- and KLCs-knockdown neurons were scored as a percentage of the numbers of accumulated Sra-1 (E) or Numb (F) in the distal part of the axon. Numbers of cells used for each calculation are more than 50, and the values shown are means ± SE of triplicates (Student’s t-test; **p < 0.01).
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Supplemental Figure 5. Effect of N-terminal fragment of DISC1 on accumulation of LIS1 and NUDEL in the distal part of the axon
A–D, Hippocampal neurons transfected with expression plasmid encoding EGFP-tagged GST (A, C) or DISC1-NB (B, D) are shown by EGFP fluorescence and staining of LIS1 (red; A, B) or NUDEL (red; C, D), and F-actin (blue). Enlarged images of the axons are shown. Bars represent 20 μm.
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Movie 1
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Movie 2
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Movie 3
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Movie 4
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Movie 5
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Movie 6
