FIGURE 4.

N-Glycosylation of AChE_T is not required for its assembly with PRiMA. A, HEK293T cells were transfected with the cDNAs encoding AChE_T^WT or AChE_T^{N296Q/N381Q/N495Q} with or without PRiMA-HA. Cell lysates containing equal amounts of AChE activity were incubated with or without an anti-HA antibody (Ab). After precipitation by protein G-agarose, the supernatants were subjected to sucrose density gradient analysis. B, cell lysates containing equal amounts of total protein were subjected to sucrose density gradient analysis. Fractions were assayed for AChE enzymatic activity and AChE_T protein by Western blotting. The upper panel shows the enzymatic activity, whereas the middle and lower panels indicate the amount of AChE_T protein in selected fractions. Sedimentation markers are shown. The enzymatic activities are expressed in arbitrary units (A.U.), and the protein is shown as % of total AChE_T protein. C, G₂ and G₄ fractions, corresponding to the shaded zones of the gradients in B, were analyzed by non-reducing electrophoresis and Western blotting with the anti-AChE antibody. Representative gradient profiles and gels from three independent experiments are shown (n = 3).