TABLE 1.
Fusion proteina | α-Adaptinb
|
γ-Adaptin
|
||
---|---|---|---|---|
Amt boundc | % Differenced | Amt bound | % Difference | |
C terminal | 657 ± 33 | NA | 618 ± 54 | NA |
Y72A | 140 ± 8 | −79 | 222 ± 19 | −64 |
Y79A | 1,009 ± 97 | +53 | 373 ± 23 | −40 |
Peripheral membrane proteins were incubated with 100 μg of fusion protein attached to glutathione beads. The fusion proteins tested had either wild-type RIDα Ile63-Leu91 sequences or Ile63-Leu91 sequences with Y72A or Y79A substitutions.
Bound proteins were immunoblotted with adaptin-specific antibodies followed by a 125I-labeled secondary antibody, and blots were exposed to a phosphor screen.
Volume integration of pixel values in a defined area with background subtracted. Pixel values are arbitrary units that are proportional to the amount of radiation stored and re-emitted from the phosphor screen. Values in the table are the means from three independent determinations for each band ± standard errors of the means and have been multiplied by 10−3. Similar results were obtained in two independent determinations.
Percent difference relative to the amount of protein bound to C-terminal fusion protein, which is set to 100%.