Effect of TCblR antiserum gene knockdown, and cDNA transfection on TC-Cbl binding. (A) Blocking of TC-Cbl binding to purified native TCblR by a polyclonal antiserum to the recombinant extracellular domain of the receptor. Apo receptor (0.1 pmol) was incubated overnight with antibody followed by measuring the binding of [⁵⁷Co]Cbl-TC (0.015 pmol) as described for Figure 3. The vertical bars represent the TC-Cbl-TCblR complex formed as determined by the Con A binding assay. The numbers in parentheses show percentage blocking in the presence of antibody. (B) Inhibition of TC-Cbl uptake in K-562 cells by anti-TCblR antibody. After incubation of 10⁶ cells with antibody for 1 hour at 4°C, binding of [⁵⁷Co]Cbl-TC (0.015 pmol) was measured for 1 hour at 4°C or 37°C. TC-Cbl binding to the receptors on the cell surface was blocked by 92% (■) when incubated with 1 μg of the antibody. Increased binding in control cells as well as cells preincubated with antiserum at 37°C represents new receptors expressed at 37°C (▧). (C) TC-Cbl uptake in CD320 knockout and wild-type ES cells. (D) TC-Cbl uptake in wild-type and TCblR cDNA-transfected HEK293 cells. For these studies, 10⁶ cells were seeded in 6-well plates for 24 hours, [⁵⁷Co]Cbl-TC (0.015 pmol) was added to 1 mL medium in the wells and incubated at 37°C for 1 hour.