Figure 5.

Palmitoylation of membrane ER46. (A) Effects of 2-hydroxymyristic acid (HMA) and tunicamycin on membrane localization of ER46. In the presence of 18 μM cycloheximide, the pretreated cells were incubated with or without (control) HMA (0.5 mM) or tunicamycin (30 μM) for 2 h. Vehicle (control) or 30 nM E2 was added for 10 min. The plasma membrane (PM) and cytosolic fractions were immunoblotted with F10 Ab. (B) Determination of palmitate or myristate incorporation into ER46. Pretreated cells were labeled in serum-free DMEM supplemented with 0.25% BSA, 100 μCi of [³H]palmitate, or 100 μCi of [³H]myristate for 4 h in the presence of 18 μM cycloheximide, followed by vehicle alone (−E2) or 30 nM E2 (+E2) treatment, subcellular fractionation, immunoprecipitation with F10 Ab, autoradiography of labeled ER for 8 weeks at −80°C, and immunodetection of total ER with F10 Ab.