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. 1997 Nov 3;139(3):601–611. doi: 10.1083/jcb.139.3.601

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Effects of the Ca²⁺-depletion protocol on the morphology and lumenal continuity of the ER as measured with erGFP. HeLa cells were transiently transfected with the erGFP construct and analyzed 2 d later. (A) ER morphology as revealed by erGFP fluorescence analyzed by confocal microscopy in live cells before (left) and 1 h after incubation in Ca²⁺-free, EGTA-containing KRB, and treatment with 30 μM tBuBHQ (right). (B) Photobleaching and recovery. After the depletion protocol was terminated, the image shown in A (control) was taken with the standard illumination protocol. A mask was then introduced in the exciting light path and the second image was taken (B; bleaching spot). The sample was then continuously illuminated with the highest laser power for 3 min before removing the mask and immediately taking the third image (C; 15 s after bleaching) with the standard settings. The last image (D; 3 min after bleaching) was collected under standard conditions, 3 min after the third. The cells were not illuminated during this recovery period. Bar, 9 μm.

Effects of the Ca²⁺-depletion protocol on the morphology and lumenal continuity of the ER as measured with erGFP. HeLa cells were transiently transfected with the erGFP construct and analyzed 2 d later. (A) ER morphology as revealed by erGFP fluorescence analyzed by confocal microscopy in live cells before (left) and 1 h after incubation in Ca²⁺-free, EGTA-containing KRB, and treatment with 30 μM tBuBHQ (right). (B) Photobleaching and recovery. After the depletion protocol was terminated, the image shown in A (control) was taken with the standard illumination protocol. A mask was then introduced in the exciting light path and the second image was taken (B; bleaching spot). The sample was then continuously illuminated with the highest laser power for 3 min before removing the mask and immediately taking the third image (C; 15 s after bleaching) with the standard settings. The last image (D; 3 min after bleaching) was collected under standard conditions, 3 min after the third. The cells were not illuminated during this recovery period. Bar, 9 μm.