Fig. 4.

IMPACT with (S)-oxoTCO–C1 reveals the PM localization of active PLD enzymes stimulated with a phorbol ester. (A) Experimental setup: endogenous PLD enzymes were stimulated with PMA (100 nM, 20 min), followed by transphosphatidylation with (S)-oxoTCO–C1 (3 mM, 5 min, in the presence of PMA), rinse (1 min), and IEDDA reaction with Tz–BODIPY (0.33 µM, 1 min), followed by confocal microscopy imaging. (B) Representative images of HeLa cells labeled as described in A with the indicated negative controls. Where indicated, PLDi (the pan-PLD inhibitor FIPI) was applied 30 min before and during the transphosphatidylation step. DKO, PLD1/2 double knockout cells. Far right: cells were rinsed after the IEDDA reaction for 20 min before imaging. White dotted lines indicate cell outlines in the negative controls. (Scale bar: 10 μm.)