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Fig. 4. Comparison of lipid rafts of untreated and G2-M-arrested Sf9 cells. Lipid rafts were isolated from unarrested and G2-M arrested Sf9 cells (lanes 2-4 and 5-7, respectively). The lipid raft fractions of G2-M cells were concentrated 30-fold after isolation. (A) Immonoblot analysis of proteins from lipid-raft- and soluble-membrane fractions probed with anti-human Cav-1 Ab. Lane 1 shows lipid raft proteins isolated from the human HT-29-MDR cell line as a control of caveolin-1 presence (Lavie et al., 1998). Lanes 2-7 represent fraction 4, 5 and 6 of the sucrose gradient that contained lipid rafts. Lanes 8 and 9 correspond to the soluble membrane fraction (fraction 11). Caveolin-1 oligomers are indicated by arrowheads. (B) Silver staining of an identical gel. Lane 1 shows protein-size marker. (C) Comparison of caveolin-immunodetected patterns of Sf9 cells (lanes 1 and 3) and human HT-29-MDR cells (lanes 2 and 4). Western blot analysis of total protein extracts incubated with anti-human Cav-1 Ab. Lanes 1 and 2 show caveolin monomers and oligomers of Sf9 cells and HT-29-MDR cells, respectively. Lanes 3 and 4 show that the caveolin bands disappear from the same blot upon probing with the same amount of anti-human Cav-1 Ab that was incubated earlier with an equivalent amount of HT-29-MDR total proteins under the same conditions to pre-absorb the anti Cav-1 Ab.
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