Fig. 1. Establishment of a stable antisense caveolin-1 HCT 116 human colorectal-carcinoma cell line. HCT 116 cells were transfected with either empty vector (control) or vector containing caveolin-1-encoding cDNA in the antisense orientation (AS-cav-1) and stable clones were selected in the presence of 0.5 mg ml^–1 puromycin for 15 days. (A) Parental, control and AS-cav-1 HCT 116 cells were solubilized in lysis buffer containing 1% Triton X-100, 60 mM octylglucoside. 20 µg protein were analysed by SDS-PAGE and immunoblotted with antibodies to human caveolin-1 and human ß-actin in order to verify equal loading. Immunoblots are shown in triplicate and are representative of at least three experiments. (B) 1 µl total RNA isolated from control and AS-cav-1 HCT 116 cells was subjected to reverse transcription followed by PCR using primer sequences to caveolin-1 and ß₂-microglobulin (as a control). PCR products were resolved on a 1.2% agarose gel stained with ethidium bromide. (C) Equal amounts of protein for HCT 116, control and AS-cav-1 cells were subjected to subcellular fractionation on a sucrose gradient after homogenization in sodium carbonate buffer, pH 11.0. Fractions were collected from the top of the gradient and equal volume aliquots of fractions 2-11 were analysed by SDS-PAGE and immunoblotted with anti-human caveolin-1 antibodies. Immunoblots are representative of at least three experiments. (D) Staining was performed on control and AS-cav-1 HCT 116 cells grown on glass coverslips. Cells were incubated in the presence of saponin with primary antibodies to rabbit anti-human caveolin-1 IgG and then incubated with Texas-Red-conjugated affinity-purified donkey anti-rabbit IgG plus normal donkey serum. The cells were observed with a Zeiss LSM 310 microscope in confocal mode at a magnification of 630 x under oil immersion. Staining for caveolin-1 is shown in red. Bar, 10 µm.