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Fig. 1. eIF4GI, eIF4E and PABP are predominantly cytoplasmic and do not directly co-localise with the actin cytoskeleton. (A) HeLa cells seeded on glass coverslips were fixed in 4% paraformaldehyde for 15 min, rinsed with PBS three times and permeabilised in PBS containing 0.8% Triton-X-100 (v/v) for 8 minutes. Rabbit antisera recognising the C terminus of eIF4GI, the N terminus of eIF4GI, eIF4E and PABP1, followed by a secondary antibody of goat anti-rabbit IgG conjugated to rhodamine, were used to visualise the localisation of the endogenous proteins within these cells (pseudocoloured in red). Actin was visualised with phalloidin-FITC (pseudocoloured green) and nuclei with DAPI (pseudocoloured blue). Scale bars: 20 µm. (B) Cells were cultured and processed as in A. The localisation of the endogenous proteins within these cells was also visualised using combinations of rabbit and mouse antisera and secondary antibodies of goat anti-rabbit IgG conjugated to rhodamine (red) or goat anti-mouse IgG conjugated to FITC (green). Left panels: rabbit anti-eIF4GI and mouse anti-eIF4E; centre panels: rabbit anti-eIF4GI and mouse anti-PABP1; right panels: rabbit anti-eIF4E and mouse anti-PABP1. White bars represent 20 µm.
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