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. 2006 Apr 24;173(2):241–251. doi: 10.1083/jcb.200506163

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Copyright © 2006, The Rockefeller University Press

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Figure 9. — Syt IV CD inhibits PC2 maturation and PC2-dependent SgII processing. (A) PC12 cells were cotransfected with HA-Syt IV CD and PC2, HA-Syt I CD and PC2, or PC2 alone. The cells were fixed and labeled with rat anti-HA (green), rabbit anti-PC2 (blue), and mouse anti-p18 (red). (B) Transfected cells were individually selected and the mean of intensity of each channel was measured. Columns represent average of the mean of intensity of each channel. Error is the SEM, where n = 22 cells for all conditions. (C) PC12 cells were cotransfected with HA-Syt IV CD and PC2. Cells positive for both HA-Syt IV and PC2 were FACS-sorted by labeling with anti-HA antibody (Alexa Fluor 488) and anti-PC2 antibody (Alexa Fluor 647). As a control, PC12/PC2 cells labeled for PC2 were FACS-sorted using identical conditions. 150,000 HA-Syt IV⁺/PC2⁺ cells and 300,000 PC12/PC2 cells were loaded on an SDS-PAGE gel and immunoblotted with the anti-PC2 antibody. 5 μg of a PC12 cell lysate was used as a negative control for the PC2 antibody. (D) PC12 cells cotransfected with HA-Syt IV CD and PC2 were fixed and labeled with anti-HA, anti-PC2, and chromogranin B (CgB) antibodies. Bars, 5 μm.