Figure 5.

Immunological cross-reaction and colocalization of antibodies raised against cubilin and antibodies against previously identified albumin binding proteins (ABP). (a) Immunoblotting using polyclonal anti-ABP antibody on affinity purified rat cubilin and rat kidney cortex. Both the IF-B₁₂ and albumin affinity purified cubilin, as well as a similar band in rat kidney cortex, were recognized by the anti-ABP. (b and c) Fluorescent double labeling reveals identical labeling using rabbit anti-ABP (1:3,000) and monoclonal mouse anti-cubilin (1:5,000) on the same section of rat cortex followed by secondary TRITC labeled anti-rabbit IgG (red fluorescent in b) and FITC-labeled anti-mouse IgG (green fluorescent in c). The antibodies colocalize along the brush border and apical cytoplasm, as well as in vesicular structures in the proximal tubules. (d and e) Inhibition of labeling with anti-ABP by preincubation with IF-B₁₂ affinity purified cubilin. Immunocytochemical labeling using anti-ABP (1:8,000) on cryosections of kidney cortex reveals proximal tubule apical labeling (d) that is clearly inhibited when the anti-ABP is preincubated with affinity purified cubilin (e). ×700.