Supplementary Information for Caudy et al., 2003-01229C
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Legend: Supplementary Figure 1. Coomassie-stained gel of sizing fractions from a RISC purification where microsequencing yielded CG7008/Tudor-SN.
Legend: Supplementary Figure 2. The sequence of Drosophila Tudor-SN. Peptides identified by MS/MS are indicated in red. Staphylococcal nuclease domains are in boldface type, the Tudor domain is underlined.
Legend: Supplementary Figure 3. Western blot analysis of Drosophila S2 cell cytoplasmic fractionation. S-100 is supernatant, P100 is pellet containing ribosomes.
Legend: Supplementary Figure 4. Fractionation of miRNAs, TSN-1, and VIG-1 in C. elegans.
A. Northern blot analysis of RNA from crude egg extract (Total), a non-ribosome associated fraction (S100) and ribosome associated fraction (S100H), probed with a DNA-oligonucleotide that recognizes the let-7 miRNA. The position of a 21nt marker is indicated on the left. Let-7 is detected in crude extract, and a large fraction of this RNA is found in the S100H fraction. Similar results are obtained with other miRNAs, like mir-40 (in eggs) and mir-52 (in adults).
B. Similar extract fractions as from a. are analysed on a western blot, and probed with VIG-1 and TSN-1 specific antibodies. Like the miRNAs, these proteins are enriched in the ribosome associated fraction.
C. Cofractionation of TSN-1 and mir-52 over resource Q and resource S columns. Only the first quarter of the gradients (0-1M) is shown.
Legend: Supplementary Figure 5. Nuclei and cytoplasm from S2 cells were fractionated via detergent lysis and western blotted for Tudor-SN. Dynein IC was used as a cytoplasmic control; histone H3 was the nuclear control.
Legend: Supplementary Figure 6. Fractionation of 293 cells into nuclear and cytoplasmic fractions via detergent lysis. Western blots were performed using anti-p100/Tudor-SN with dynein IC as a cytoplasmic control and histone H3 as a nuclear control.
Legend: Supplementary Figure 7. Immunofluorescence analysis of Tudor-SN in Drosophila S2 cells. Control is without primary antibody. Primary antibody is affinity-purified rabbit antibody directed against the C-terminus of Tudor-SN. DAPI merge is shown below primary only.