Figure 2.

GalNAc-T and Gal-T2 interact physically. (A) Immunoprecipitation (ip) of lysates of clone 3 transiently transfected with an HA-tagged Gal-T2 was performed by using either anti-c-myc (lanes 1 and 5) or anti-HA (lanes 2 and 4) antibodies. Immunocomplexes were analyzed by SDS/PAGE and Western blotting using anti-HA or anti-c-myc antibodies, as indicated. Lanes 3 and 6 correspond to a mixture of lysates from the single transfectant clones 3 and IGT2 (induced with 1 μM ponasterone) carrying enzyme activities comparable to those present in the double transfectants (specific activity, 1 and 2 nmol × mg prot⁻¹ × h⁻¹ of, respectively, GalNAc and Gal transferred to the corresponding exogenous acceptors) and subjected to the whole immunoprecipitation procedure. (B) GalNAc-T does not coimmunoprecipitate ManII. Lysates of clone 3 were immunoprecipitated with anti-ManII (lanes 1 and 3) or anti-c-myc (lanes 2 and 4) antibodies and Western blotted with anti-ManII or anti-c-myc antibodies, as indicated. Three independent experiments were run, which gave essentially the same results.