FIGURE 7.

N-Glycosylation is not required for the formation of human-chicken AChE dimers and tetramers. A, HEK293T cells were transfected with equal amounts of cDNAs encoding human AChE_T or AChE_T^{N296Q/N381Q/N495Q} together with chicken AChE_T (cAChE_T). Equal amounts of protein from cell lysates were subjected to sucrose density gradient analysis. The enzymatic activities are plotted as a function of the S value, estimated from the position of sedimentation markers (upper panel). G₂ fractions were collected for immunoprecipitation (IP) by either anti-human AChE antibody (Ab) or anti-cAChE antibody. The immunoprecipitated complexes were analyzed by SDS-PAGE and Western blotting with anti-AChE and anti-cAChE antibodies, as indicated (lower panel). Both antibodies did not cross-react with other corresponding antigen. B, HEK293T cells were transfected with equal amount of cDNAs encoding AChE_T or AChE_T^{N296Q/N381Q/N495Q} together with cAChE_T and PRiMA. Equal amounts of protein from cell lysates were subjected to sucrose density gradient analysis as in A. G₄ fractions were collected for immunoprecipitation as in A. C, extracts from cells expressing AChE_T^WT and cAChE_T separately or together were analyzed by non-reducing SDS-PAGE and labeled with anti-cAChE and anti-AChE antibodies. The presence of an intermediate band between cAChE and AChE_T^{N296Q/N381Q/N495Q} dimers, recognized by both antibodies, demonstrates the formation of mixed cAChE_T-AChE_T^{N296Q/N381Q/N495Q} dimers. IB, immunoblot.