Figure 1

Three types of secretion for Gaussia luciferase activity. A, Constructs used in this study are wild-type Gaussia luciferase (GLUC), an N-terminal deletion mutant that has no signal peptide (dNGLUC), GFP-tagged GLUC with an internal signal peptide (GFP-_SPGLUC), GFP-tagged dNGLUC and β-actin-tagged GLUC with and without an internal signal peptide (Actin-_SPGLUC, Actin-dNGLUC). The signal peptide is shown as a black box, GLUC without signal peptide is shown in white, GFP as a striped box and β-actin as a grey box. B, 293 T cells were transfected with dNGLUC, GFP-dNGLUC and β-actin-dNGLUC, supernatants and cell lysates were collected after 24 h and analyzed for luciferase activity. GLUC activity was measured as described in Methods. C, 293 T cells were transfected with dNGLUC and GFP-dNGLUC and cultured for 24 h prior to a media change. Fresh medium containing DMSO or 10 μg/mL Brefeldin A was added to the cells and supernatants were collected after 2 h for analysis of luciferase activity. D, 293 T cells were transfected with GLUC, GFP-_SPGLUC and β-actin-_SPGLUC and analyzed for luciferase activity in supernatants after 24 h. E, 293 T cells were transfected with GFP-_SPGLUC and βActin-_SPGLUC, treated with Brefeldin A and supernatants were collected and analyzed for luciferase activity as described. B-D, Shown is the mean of three independent experiments with standard error bars. R.L.U. – relative light units, SN – supernatant.