Figure 6.

Stable reexpression of wild-type Munc18-1, but not K46E/E59K mutant, restores syntaxin-1 expression and NE-secretion in Munc18-1/-2 double knockdown clones (D7 and D16). (A) Munc18-1/-2 double knockdown clones (D7 and D16) were infected with lentiviruses that express EmGFP, wild-type Munc18-1-EmGFP or mutant (K46E/E59K) Munc18-1-EmGFP, and the infected cells were selected with blasticidin. The surviving cells were further enriched by a FACS using the GFP signal. For the wild-type Munc18-1-EmGFP, we separated two populations of cells in which the GFP level differs (high vs. low). Thirty micrograms of total homogenates from heterologous pools of the enriched cells was analyzed by SDS-polyacrylamide gel electrophoresis and immunoblotting using anti-Munc18-1 and syntaxin-1 antibodies. Signals were detected with enhanced chemiluminescence detection system. A number on the left indicates the position of a molecular weight marker. (B and C) NE release was stimulated by 70 mM KCl for 15 min in the rescued cells (B for D7 clones; C for D16 clones). Error bars indicate SEM (n = 15).