Figure 7: In vitro COPII budding reaction supported by wildtype or TRAPPC12−/− cytosol.
From: Mammalian TRAPPIII Complex positively modulates the recruitment of Sec13/31 onto COPII vesicles
(A) Schematic drawing of the procedures of in vitro COPII budding assay. (B) A representative experiment of COPII budding reaction. ER membranes from wildtype HeLa cells were mixed with no cytosol (lanes 1 and 4), wildtype cytosol (lanes 2 and 5), or TRAPPC12−/− cytosol (lanes 3 and 6) to allow COPII vesicles budding to occur. 10% input and 100% of budded vesicles were loaded on SDS-PAGE. (C) Quantitative analysis of COPII budding assay. Sec31A- and Sec23A-specific bands were quantified by ImageJ software. The budding efficiencies were 3.08% (Sec31 WT), 0.68% (Sec31, C12 KO), 7.64% (Sec23 WT), and 7.62% (Sec23, C12 KO) of the corresponding input signals (i.e., comparing signals of lane 5 to lane 2, and lane 6 to lane 3). The data were shown Mean ± S.D., n = 3, P < 0.01. (D) COPII budding reactions were carried using ER template membranes isolated from a HeLa cell line stably transfected with SEAP as transport cargo. The budded COPII vesicles were measured with alkaline phosphatase activity as an indication of cargo transport. The data represented averages of above-background alkaline phosphatase activities of the two samples, using no cytosol control as background. Error bars = S.D., n = 3, P < 0.01.
