FIGURE 3:

Palmitoylation of ELMOD2. (A) GFP-ELMOD2 cDNA or PSD-95 cDNA was transfected to HEK293T cells with HA-DHHC3 cDNA or HA-GST cDNA. After metabolic labeling with [³H]palmitate, proteins were separated by SDS–PAGE, which was followed by autoradiography (top panel). Western blotting (IB) for GFP, PSD-95, and HA is shown (bottom panels). When DHHC3 cDNA was cotransfected, [³H]palmitate was incorporated to GFP-ELMOD2 (arrowhead), under the conditions PSD-95 was palmitoylated (arrow). (B) cDNA of GFP alone or that of various GFP-ELMOD2 constructs was transfected to HEK293T cells with either HA-DHHC3 cDNA or HA-GST cDNA. Autoradiography and Western blotting were detected as described in A. GFP-ELMOD2 proteins incorporating [³H]palmitate are shown by an arrowhead. (C) Huh7 cells expressing either GFP-ELMOD2 (WT) or GFP-ELMOD2 (−5Cys) were lysed and examined by the ABE method for palmitoylation. Only the lysate of the GFP-ELMOD2 (WT)-expressing cell showed intense labeling at the expected molecular mass, indicating palmitoylation of GFP-ELMOD2 in cells.