Fig. 2. The pH-dependent internalization and binding of IgG is impaired in the absence of ß₂m. (A) IgG internalization. FO-1 cells (a,e), FO-1 ß2m cells (b,f), FO-1 cells expressing Myc-hFcRn (c,g), and FO-1 ß2m cells expressing Myc-hFcRn (d,h) were incubated in medium at pH 6.5 (a-d) or pH 7.4 (e-h) in the presence of hIgG (1 µg/ml) for 30 minutes at 37°C. After cooling the cells on ice and washing off unbound IgG, cells were fixed, permeabilized and stained with labeled secondary antibodies to detect internalized hIgG. (B) Binding of FcRn to IgG-agarose. FO-1 cells (lanes 1,5), FO-1 cells expressing Myc-hFcRn (lanes 2,6), FO-1 ß2m cells (lanes 3,7) and FO-1 ß2m cells expressing Myc-hFcRn (lanes 4,8) were lyzed in CHAPS buffer at pH 6.5 (top panels; pH 6.5) or pH 7.4 (bottom panels; pH 7.4). Cell lysates were blotted using monoclonal anti-Myc (-Myc) or polyclonal anti-ß₂m (-ß2m) antibodies (lanes 1-4; lysate). Alternatively, cell lysates were incubated with IgG-agarose and bound proteins then eluted and blotted (lanes 5-8; IgG binding). The data in Fig. 2 is representative for at least three independent experiments carried out using two different cell clones.