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. 2010 Oct 4;285(49):38710–38719. doi: 10.1074/jbc.M110.137414

FIGURE 5.

FIGURE 5.

CAPS1 knockdown induces chromogranin accumulation in the Golgi complex. A, KD of CAPS1 expression in PC12 cells by siRNA. siRNA specific for CAPS1 was electroporated into PC12 cells, and the culture medium was concentrated and electrophoresed. Top, three bands were greatly reduced in the culture medium of the CAPS1 KD cells and were identified as chromogranin A (ChgA), chromogranin B (ChgB), and secretogranin II (SgII) by LC-MS/MS. Bottom, reduction of CAPS1 expression in PC12 cells by siRNA KD was assayed by immunoblotting of cell lysates with anti-CAPS1 antibody. B, high KCl-induced chromogranin A and secretogranin II release from CAPS1 KD PC12 cells. PC12 cells were transfected with either control siRNA or CAPS1 siRNA together with a ChgA-HA or SgII-HA expression plasmid. The culture media, treated with 5 mm KCl (5K) or 50 mm KCl (50K) (see “Experimental Procedures”), were collected and analyzed by immunoblotting with anti-HA antibody. C, statistical analyses of the effect of CAPS1 KD on ChgA-HA release from PC12 cells. PC12 cells were transfected with ChgA-HA together with the control (white bar) or CAPS1 siRNA (black bar). The amounts of ChgA-HA released into the culture media with 5 or 50 mm KCl stimulation were analyzed by Western blotting, followed by densitometric analysis (n = 6). There was no significant difference in the amount of ChgA-HA in cell lysates between the control and CAPS1 KD groups (n = 6). The signal intensities of the extracellular ChgA-HA bands of the culture media were normalized against those of the intracellular ChgA-HA bands of the cell lysates. AU, arbitrary unit. Error bars, S.E. **, p < 0.01 by Student's t test. D, subcellular localization of endogenous chromogranin A in NGF-differentiated PC12 cells without (left) and with (right) CAPS1 KD; immunostaining for chromogranin A. Scale bars, 20 μm. E, subcellular localization of ChgA-HA expressed in NGF-differentiated PC12 cells without (left) and with (right) CAPS1 KD; immunostaining for HA. Scale bars, 20 μm. F, subcellular localization of C-terminal HA-tagged TrkB expressed in NGF-differentiated PC12 cells without (left) and with (right) CAPS1 KD; immunostaining for HA. Scale bars, 20 μm.