No or little effect of CHC833-1406 expression on clathrin-mediated endocytosis. (a) Expression of CHC833-1406 does not affect transferrin receptor-mediated endocytosis. HeLa cells were transfected with indicated plasmids plus a green fluorescent protein (GFP) expression plasmid. Three days after transfection, cells were incubated for 8 min at 37 °C in DMEM containing 20 μg ml−1 of AlexaFluor-594-conjugated transferrin (AF594-transferrin) and 0.1% BSA. After removal of cell surface-bound AF594-transferrin, these cells were trypsinized and fixed with 4% paraformaldehyde. For measurement of AF594-transferrin uptake, GFP-positive cells were quantified by flow cytometric analysis as described in Materials and methods. (b) Analysis of the expression of transferrin receptors on cell surface. HeLa cells were transfected as described in (a), detached with 1 mM EDTA/PBS and incubated on ice for 20 min in DMEM containing 20 μg ml−1 of AF594-transferrin and 0.1% BSA. After washing with ice-cold PBS, these cells were fixed and the amount of AF594-transferrin bound on cell surface was quantified by flow cytometric analysis. (c) Expression levels of CHC and transferrin receptor in HeLa cells transfected with pSUPER-CHC and CHC expression vectors. Expression levels of CHC and transferrin receptor in cell lysates from cells transfected as described in (a) were analysed by immunoblotting with anti-CHC (X.22) or antitransferrin receptor antibody for confirmation. The amount of β-tubulin is indicated as a loading control.