FIGURE 4.

GluR6 S846D and GluR6 S868D are more highly retained in the ER than GluR6 WT. HeLa cells were transfected with FLAG-GluR6 (WT, S846A, S846D, S868A, or S868D). Cell lysates were prepared and subjected to a glycosidase assay as described under “Experimental Procedures.” Proteins were immunoblotted with GluR6 antibody. A, GluR6 S846D and GluR6 S868D were more endo H sensitive than GluR6 WT, GluR6 S846A, or GluR6 S868A. Endo H-sensitive fractions were quantitated by measuring the band intensity of the endo H-sensitive fraction compared with the band intensity of endo H-resistant fraction using NIH Image software. Error bars indicate S.E.M. *, p < 0.05, Student's t test. B, PKC activation increases ER retention of GluR6. Twenty-four hours after transfection, cells were treated with PMA (1 μm) and MG132 (20 μm) for the indicated time. GluR6 from PMA-treated cells was more endo H sensitive, as detected by a change in the ratio of immature GluR6 (lower band) to mature GluR6 (upper band) following endo H treatment. WB, Western blot.