GRA7-III-induced activation of PLD1 was dependent on interaction with PLD1.
(A) Bacterially purified 6xHis-GRA7-III and its mutants were analyzed by Coomassie blue staining (left) or IB with αHis (right). (B) BMDMs from PKCα+/+ and PKCα-/- mice were stimulated with rGRA7-III (5 μg/ml) and its mutants for the indicated times, followed by IB to detect phosphorylated and total forms of PLD1 and PLD2. Actin was used as a loading control. (C) BMDMs were stimulated with rGRA7-III and its mutants for 30 min. and analyzed for PLD activity assay in vitro. Data shown are the means ± SD of five experiments. (D) Fluorescence confocal images in BMDMs were stimulated with rGRA7 and its mutants for 30 min., fixed, immunostained with antibodies for His (Alexa 488) and PLD1 (Alexa 568). Scale bar, 20 μm. The data are representative of three independent experiments with similar results (A, B, and D).