FIGURE 6.

NPY secretion defects in Munc18-1 knockdown PC12-KD43 cells are rescued upon reintroduction of Munc18-1 (WT and the indicated mutants) by transfection. A, immunoblot analysis of transfected Munc18-1 proteins in KD43 cells. 30 mg of total homogenates from KD43 cells electroporated with 20 μg of the empty plasmid (pCMV5 as control) or the plasmid that expresses Munc18-1 proteins were analyzed by SDS-PAGE and immunoblotting using anti-Munc18-1 and anti-VCP/p97 antibodies. VCP/p97, a ubiquitous membrane trafficking protein (42), was used as a loading control. B, secretion of transfected NPY-hPLAP from the knockdown (KD43) cells that were co-transfected with the control plasmid or the Munc18-1-WT, Munc18-1-F115E, or Munc18-1-E132A expression plasmids (n = 16). C, secretion of NPY-hPLAP from the KD43 cells that were transfected with the control plasmid or the Munc18-1-WT or Munc18-1-F115E/E132A expression plasmids (n = 15). D, average secretion of transfected NPY-hPLAP from 5 different clones of control PC12 cells (pSuper) that were co-transfected with the control plasmid. Data were expressed as mean ± S.E. and considered significant at * p < 0.05. ns, not significant.