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Figure 4


Fig. 4. Enlargeosome exocytosis in PC12-27 cells that were microinjected with antibodies. (A-D) [Ca2+]i and surface-capacitance traces in patched-clamped cells. Photolysis of the NP-EGTA caged Ca2+ compound induced similar [Ca2+]i rises in non-microinjected (A) and microinjected (B-D) cells (bottom traces). The capacitance surface-expansion responses (upper traces) were similar in non-microinjected cells (A) and in cells microinjected with monoclonal anti-ChgB antibody; ChgB is not expressed by PC12-27 cells. (B) By contrast, these responses were greatly reduced in cells that were microinjected with antibodies against SNAP23 (C) and annexin-2 (D). The time and % capacitance increase bars in A are valid also for B-D. (E) Averages ± s.d. of the capacitance-increase data in groups of 10-15 cells. (F-M) The enlargeosome exocytic responses, revealed by surface immunolabeling by desmoyokin (red), that were induced by ionomycin treatment (3 µM, 5 minutes) in cells microinjected with anti-ChgB, anti-SNARE or anti-annexin-2 antibodies (labelled in green) are shown. The cells that exhibit no green labelling were non-microinjected. Enlargeosome exocytoses were approximately the same in non-microinjected cells and in those microinjected with antibodies against ChgB (F), VAMP7 (J), VAMP8 (K) and Stx4 (L); they were greatly reduced in the cells microinjected with antibodies against SNAP23 (G), annexin-2 (H), VAMP4 (I) and Stx6 (M). Scale bar: 5 µm. (N) Averages ± s.d. of the data obtained for the immunolabeling of surface desmoyokin in groups of 18-22 cells stimulated with ionomycin. The indications specified in the bars refer to the microinjected antibodies. (O) Averages ± s.d. of the data obtained for the immunolabeling of surface desmoyokin (d/A) in groups of 18-22 cells, resting (NT) or stimulated with ionomycin (iono) without or with pre-treatment with botulinum toxin E (BtxE).





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