Figure 8.
Munc13-4 is a positive regulator of degranulation. Western blot of lysates prepared from stable RBL-2H3 transfectants expressing GFP, YFP-Munc13-4, GFP-Munc13-4(aa 543-1090), and Munc13-4Δ608-611. Expressed constructs were detected with antibodies against Munc13-4, and tubulin (loading control). Arrowhead denotes endogenous Munc13-4, open arrow YFP-Munc13-4 and GFP-Munc13-4Δ608-611, and closed arrow GFP-Munc13-4(aa 543-1090). Transfectants were stimulated with IgE/TNP-bovine serum albumin, and degranulation was measured as release of β-hexosaminidase into the medium. Results are calculated as percentage of total β-hexosaminidase present in the cells. Nonspecific release of β-hexosaminidase was measured in the absence of TNP-bovine serum albumin and was <3% for the four cell lines. Moderate overexpression of YFP-Munc13-4 enhanced secretion significantly above control GFP-expressing cells (Student's t test, p <0.005). Data are means of quadruplicates ± SE.