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Fig. 1. Depletion of tGolgin-1 from HeLa cells using siRNA. (A) The mRNA encoding tGolgin-1, showing the positions of the overlapping 21-mers used for siRNAs relative to the coding region and the 5' and 3' untranslated regions. See Table 1 for the sequences of the oligonucleotides. (B,C) Low-magnification (40x) IFM image of tGolgin-1 (B) and TGN46 (C) expression in HeLa cells transduced once 2 days earlier with siRNA-1. Cells were co-stained with antibodies against tGolgin-1 and TGN46, and with appropriate fluorochrome-conjugated secondary antibodies. Arrows denote cells with reduced tGolgin-1 expression. (D) Untransfected HeLa cells (control) or cells transduced once with siRNA-1 were analysed by IFM as in B,C. Cells were counted as positive or negative for tGolgin-1 expression as indicated and, within each population, cells were scored for a `normal' ribbon-like or a dispersed, punctate staining pattern for TGN46 in single plane images. The proportions of cells in each category are given from a representative experiment. Results with two control siRNAs and with siRNA-4 were similar to those obtained with untransfected cells (E). Whole-cell lysates from HeLa cells transduced twice with siRNA-1 or control siRNA were fractionated by SDS-PAGE and analysed by western blot for tGolgin-1 production. Only the portion of the gel corresponding to full-length tGolgin-1 (260 kDa) is shown; the rest of the lanes were blank.

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