Figure 1.

Gel filtration of HeLa cell extract containing cytoplasmic and plasma membrane proteins. A. The preparation of Triton-X100 soluble cell extract was analyzed by gel filtration using a superose-6 column. One ml fractions (numbered from 1 to 25) were collected after the elution of the void volume (Vo), and the presence of nucleolin was monitored by immunoblotting using rabbit polyclonal antibodies against the NH₂-terminal peptide of nucleolin. The numbers 232, 110 and 67 on the top indicate the position of protein markers of molecular weight 669-, 232-, 110-, and 67-kDa. NCL on the right shows the main nucleolin band at position 100-kDa. The other low molecular weight bands represent the cleaved products of nucleolin. B. HeLa cells were first incubated with 5 μM of HB-19/Btn at room temperature before preparation of cell extracts. Gel filtration was carried out as in section 1A, but only one every other sample was analyzed by immunoblotting. C/D. The peak of nucleolin eluting at an apparent molecular weight of 500-kDa is complexed with HB-19/Btn. Fractions 1-7 and 17-24 from the gel filtration experiment described in section B were purified by affinity chromatography using Avidin agarose and analyzed by immunoblotting. On the right of the different gels is the position of the molecular weight markers. All experimental procedures were as described in Methods.