The Journal of Neuroscience, January 26, 2005, ():
Differential Transport and Local Translation of Cytoskeletal, Injury-Response, and Neurodegeneration Protein mRNAs in Axons
J. Neurosci. Willis et al.
25: 778
Supplemental data
Files in this Data Supplement:
- supplemental material
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Supplemental Figure S1: Fractionation of axonally synthesized proteins on pH 3-10 2D gel.
Broader pH range IPG strips were used to evaluate synthesis of more basic proteins in the axons. Representative images from 2D gels from analytical pH 3-10, 11 % SDS/PAGE fractionations of 35S-labeled axonal preparations (A, B) and matching preparative fractionations from whole DRG culture lysates (C) are shown. A shows the image of radioactive proteins by phosphorimaging; B shows the aurodye stain for total proteins in blot from panel A. C shows preparative gel of whole DRG lysates that was stained with Coomassie. The numbered spots indicate proteins that could be aligned between the gels. These spots were excised from the preparative gel and processed for MS analyses (see Table 2).
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Supplemental Figure S2: Confirmation of MS results by RT/PCR.
Cell body andaxonal RNAs were isolated as outlined in Materials and Methods and used for RT/PCR. Negative control consisted of axonal RNA processed for reverse transcription and PCR without addition of reverse transcriptase (no RT). The primer pairs used for amplification are detailed in Table 1. (ATP synthase = ATP synthase subunit d; CsA = peptidyl isomerase A/cyclophilin A; Ddah 2 = dimethylarginine dimethylaminohydrolase 2; GAPDH = glyceraldehyde 3-phosphate dehydrogenase; PGK 1 = phosphoglycerate kinase 1; Prdx 1 = peroxiredoxin 1; Prdx 6 = 1-cys peroxiredoxin).
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Supplemental Figure S3: Quantitative PCR analyses of axonal mRNA transport after local neurotrophin treatment.
Real-time RT/PCR was used to quantitate levels of the indicated axonal mRNAs after 4 h treatment of axonal compartment with BSA-, NGF- or BDNF-absorbed microparticles. RNA templates were normalized to protein content. Quantitative PCR signals for individual templates were further normalized to signals for 12S mitochondrial rRNA and are expressed relative to the BSA-treated axonal preparations.
