Photoactivation experiments were conducted as previously described (35). PA-GFP imaging was performed in multitracking mode on a Zeiss LSM 880 NLO laser scanning confocal microscope (Carl Zeiss, Thornwood, NY) with a 63× Plan Apochromat 1.4 NA objective and a 413/488 dichroic mirror. In brief, pPAGFP-N1 or pPAGFP-N1-CerS1 plasmids were cotransfected with pCMV6-AC-CalR-mRFP-ER in HeLa cells. After 48 hours, post-transfection cells were loaded with MitoTracker Far Red (Invitrogen) for 45 min. Immediately before imaging, 5 μM SoSe was added to the cells. After three initial frames, cells were irradiated by high energy (1 μW) of 405 laser light. Images were shot at 5 and 90 min after photoactivation.

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