Figure 6

Release of Gaussia luciferase is suppressed by GGA1. A, To test efficiency of shRNA mediated GGA knockdown 293 T cells expressing Flag-tagged GGA1 were co-transfected with pLKO control vector or pLKO shGGA. Cell lysates were resolved on 10% PAGE and transferred to PVDF membranes. The blots were probed with anti-Flag M2 or anti-β-actin antibody. B, 293 T cells were co-transfected with dNGLUC and the shRNA vectors pLKO (control) or pLKO shGGA and cultured for 72 h. Supernatants were harvested and analyzed for Gaussia luciferase activity using a PerkinElmer TopCount. C, 293 T cells were co-transfected with dNGLUC and an expression vector for GFP or GGA1 and cultured for 24 h prior to analysis of GLUC activity in supernatants as in A. D, FGF2 was transfected into 293 T cells together with a control vector (pLKO) or an shRNA vector targeting GGA1. FGF2 secretion into supernatants was measured using an ELISA kit (R&D Systems) after 24 h. E, 293 T cells expressing dNGLUC were co-transfected with pLKO vector control or shGGA and wild-type Flag-GGA1 or a mutant version of GGA1 that is refractory to shRNA mediated knockdown by shGGA (GGA1m). Supernatants were harvested after 48 h and analysed for GLUC activity as previously described. B-E, shown are the results from three independent experiments plus standard deviation. R.L.U. – relative light units, SN - supernatant.