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. 2009 Dec 1;20(23):4962–4975. doi: 10.1091/mbc.E09-08-0712

Figure 2.

Figure 2.

Munc18-1/-2 double knockdown clones exhibit significant reductions in the expression of all the plasma membrane syntaxins (syntaxin-1, -2, and -3) and abolish regulated NE secretion. (A) Immunoblot analysis of isolated multiple clones in which both Munc18-1 and -2 are strongly down-regulated. Thirty micrograms of total homogenates from the Munc18-1/-2 double knockdown (D) and control (C) clones was analyzed by SDS-polyacrylamide gel electrophoresis (PAGE) and immunoblotting using anti-Munc18-1 and -2; syntaxin-1, -2, and -3; SNAP-25; and VCP/p97 (as a loading control) antibodies. Signals were detected with enhanced chemiluminescence detection system. A number on the left indicates the position of a molecular weight marker. (B) Quantification of changes in protein expression of syntaxin-1, -2, and -3; SNAP-25; and VCP between control and Munc18-1/-2 double knockdown clones. Images of these proteins on the film were quantified using ImageJ. **p < 0.01), statistically significant difference. (C) NE release was stimulated by 70 mM KCl for 15 min. Seven pairs of control and double knockdown clones were examined. Error bars indicate SEM (n = 6–12).