FIGURE 2.

Expression and ¹²⁵I-HA ligand binding of 190-HARE CD mutants. Cells were grown to confluence, washed twice with cold PBS, and centrifuged cell lysates (25 μg of protein) were subjected to SDS-PAGE and electrotransfer. A, HARE protein was detected by Western blot analysis using anti-V5 Ab. The 190-HARE proteins are as follows: lane 1, WT; lane 2, ΔM1; lane 3, ΔM2; lane 4, ΔM3; lane 5, ΔM4; lane 6, ΔM1M2; lane 7, ΔM1M2M4; lane 8, Y2519A; and lane 9, EV. B, ligand blot assays were performed with ¹²⁵I-HA in multiple experiments using different clones of the indicated mutants, and HARE expression was determined by Western blotting using the same strips after autoradiography. ¹²⁵I-HA ligand blot signals were normalized to HARE expression using densitometry and to WT (as 100%).