Fig 4.

The lumenal domain of A33 is sufficient for interaction with B5-GFP. (A) Diagrammatic representation of constructs used for CoIP. Soluble B5 containing the signal peptide (SP), the lumenal domain (LD), and the coiled-coil (cc) domain of B5 (▨). Soluble A33-HA containing the cleavable signal peptide (SP) sequence of VSVG (□), followed by the predicted lumenal domain (LD) of A33 (▨) and the HA epitope tag. Connecting domains that were deleted are represented by a dashed line in the diagram (B). CoIP. HeLa cells infected with vTF7.3 in the presence of AraC were transfected with either the indicated plasmids that expressed the constructs described in panel A or pCDNA3 (Empty Vector). At 4.5 h posttransfection, the medium was replaced with medium containing [³⁵S]Met-Cys. Cells were harvested at 24 h posttransfection and lysed. Cell lysates were immunoprecipitated with an anti-HA MAb, immune complexes were resolved by SDS-PAGE, and proteins were detected by autoradiography. (C) Protein expression. Cell lysates used in panel B were resolved by SDS-PAGE and analyzed by Western blot assay using an anti-B5 (top) or anti-HA (bottom) MAb, followed by an HRP-conjugated donkey anti-rat antibody. The molecular masses, in kilodaltons, and positions of marker proteins are shown on the left of the blots.