(A) Expression level of UPIIIa in 5637 and some other human cancer cell lines. (B) 5637 cells were cultured in serum-containing medium (FCS +), serum-free medium in the absence (FCS −) or the presence of 15 µM MβCD (FCS −, MβCD). Cell samples were subjected to subcellular fractionation by discontinuous sucrose density gradient ultracentrifugation as in Fig. 3D, and fractions obtained were analyzed by immunoblotting with the anti-UPIII antibody. The positions of the MD and the non-MD fractions were indicated by opened squares. Asterisks indicate the positions of the 45-kDa UPIIIa. (C) Triton X-100-solubilized cell extracts were prepared from 5637 cells that had been exposed to serum-free medium for the indicated times (0–48 h). Protein samples (30 µg/lane) were analyzed by immunoblotting with anti-UPIII antibody. Closed and opened arrowheads indicate the positions of the 45-kDa UPIIIa and its degraded fragment, respectively. (D) Serum-starved 5637 cells (24 h of serum starvation) were treated with serum-containing medium for the indicated times (0–8 h). Closed and opened arrowheads indicate the positions of the 45-kDa UPIIIa and its degraded fragment, respectively. In panels C and D, control data obtained with the normally-cultured 5637 cells (FCS) are also shown.