The Journal of Neuroscience, May 31, 2006, ():
Regulation of Intracellular Trafficking of Huntingtin-Associated Protein-1 Is Critical for TrkA Protein Levels and Neurite Outgrowth
J. Neurosci. Rong et al.
26: 6019
Supplemental data
Files in this Data Supplement:
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Figure S1 Effect of phosphorylation on HAP1A and its association with KLC and p150 dynactin. (A) GST fusion proteins containing full-length KLC-2 (GST-KLC) or p150 dynactin (amino acids 1023-1223, GST-p150) were incubated with HEK cell lystates containing transfected HAP1A or T598A. Purified GST, GST-KLC, and GST-p150, which were used for in vitro binding, are revealed by western blotting with anti-GST (left panel). The precipitates were analyzed by western blotting with the mouse antibody to HAP1 that reacts with both HAP1A and T598A (right panel). Note that GST-KLC and GST-p150 bind more T598A than wild type HAP1A. (B) Densitometry analysis of the ratios of bound proteins to input from 4 experiments.
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Figure S2 Adenoviral infection of PC12 cells with scramble siRNA and GFP or HAP1 siRNA. (A) Fluorescent images showing that HAP1 siRNA, but not the control scramble siRNA, inhibits neurite extension of PC12 cells. Two days after adenoviral infection, GFP (green) is also expressed in adenoviral infected cells. (B) Densitometry analysis of the ratios of HAP1, TrkA, and p75NTR receptor to tubulin on western blots, which reflect the relative change of these proteins at their levels, in PC12 cells that had been infected with the scramble siRNA or HAP1 siRNA. The data (meanąSE) were obtained from 3 experiments. ** p<0.01.
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Figure S3. Adenoviral infection of cultured sympathetic neurons isolated from the superior cervical ganglia in adult (3-4 weeks old) rats. Adenoviral HAP1 siRNA vector, which also expressed GFP, infected both cultured glial cells and HAP1-containing sympathetic neurons (arrows and red). The control neurons were infected with adenoviral scramble siRNA and GFP. Note that HAP1-positive neurons (red) show extended neurites (indicated by stars) in the control sample. HAP1 siRNA infection, however, reduced the neurite length of cultured neurons.
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Figure S4 Mutant huntingtin does not affect the phosphorylation of HAP1A. (A) Western blots of PC12 cells that had been infected by adenoviral GFP-htt-23Q or GFP-htt-130Q for 2 days. Endogenous HAP1 and GFP-htt were analyzed by western blotting with antibodies to p1A, HAP1A, and htt (mEM48). Two samples of each group are shown. (B) Western blotting of the whole brain tissues from wild type (WT) and N171-82Q transgenic mice at the age of 2.5 months. Results from 2 mice each group are shown here. Brain tissues from HD 150CAG knock-in (KI) and wild type mice at 2 months of age were also analyzed by western blotting.
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Supplementary movies PC12 cells were transfected with RFP-HAP1A and then infected by GFP-htt-23Q or GFP-htt-130Q for 16 h. The cells were treated with NGF (100 ng/ml) for 36 h to examine the distribution and movement of these proteins in neurites. The merged images were shown, in which colocalization of GFP-htt (green) and RFP-HAP1A (red) yields yellow color. Note that htt-23Q infected cells show more HAP1A puncta in the neurites and tips, which move biodirectionally. Compared to htt-23Q cells, fewer HAP1A puncta are present in the neurites of GFP-htt-130Q infected cells. The htt-HAP1 puncta in GFP-htt-130Q cells also move slowly as compared with those in htt-23Q cells. Note that in GFP-htt-130Q transfected cells (upper one), htt inclusions sequester RFP-HAP1A and become larger during the course of imaging. The movies consist of 50 images that were captured every 25 seconds over about 20 minutes.
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Supplemental movie 2
