FIG 3.

Posttranslational palmitoylation targets LpdA to membranes. (A) Putative C-terminal palmitoylation site of LpdA. The three cysteines predicted to be modified with a palmitic acid moiety are underlined. (B) HA-LpdA, HA-LpdA C440F, and HA-LpdAΔC5aa were ectopically expressed in HeLa cells and immunostained, and their localization was analyzed by IF microscopy. Shown are representative images of at least three independent experiments. Scale bar, 10 μm. (C) Metabolic labeling and protein isolation strategy. HeLa cells expressing LpdA were metabolically labeled with the palmitic acid analogue ODYA. After cell lysis, a biotin moiety and a fluorophore were ligated using click chemistry. Posttranslationally modified proteins were isolated using streptavidin pulldown (PD) and analyzed by Western blotting (WB). (D) Representative result of the WB analysis of PD and input fractions using HA-conjugated, calnexin (endogenous metabolic labeling control), and tubulin (separation control) antibodies.