Fig. 3. Expression and localization of p11 to caveolae are reduced in AS-cav-1 HCT 116 cells. (A) 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 p11 and ß2-microglobulin (as a control). PCR products were resolved on a 1.2% agarose gel stained with ethidium bromide. (B) Control and AS-cav-1 HCT 116 cells were solubilized in lysis buffer containing 1% Triton X-100 and 60 mM octylglucoside. 25 µg protein were analysed by SDS-PAGE and immunoblotted with anti-human-p11 antibody. Immunoblots are shown in triplicate and are representative of at least three experiments. (C) Equal amounts of protein for control and AS-cav-1 HCT 116 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-p11 antibodies. Immunoblots are representative of at least three experiments.