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Figure S2. The loss of mBet3p blocks COPII vesicle tethering and disrupts Golgi architecture. (A) mBet3p is also found on endosomal structures. The arrow points to mBet3p (small gold particles) on tubules connected to a multivesicular body. (B) Excess purified His₆-mBet3p completely neutralizes the inhibition of anti-mBet3p antibody. Two COPII vesicle populations were formed in vitro using permeabilized NRK cells in a stage I incubation, as described in Fig. 5. The two vesicle populations were then combined and preincubated together for 20 min on ice in the absence or presence of antibody and/or excess purified His₆-mBet3p. Subsequently, one sample was kept on ice, while the other samples were incubated at 32°C for 60 min in a stage II incubation before the addition of anti-Myc antibody and quantitation of heterotrimeric VSV-G* in the precipitate. 1, ice; 2, one population of vesicles that was untagged instead of Myc-tagged; 3, complete reaction incubated without antibody; 4, 275 nM α-mBet3p antibody; 5, 275 nM α-mBet3 antibody plus 13.75 µM His₆-mBet3p; and 6, complete reaction with 13.75 µM His6-mBet3p. (C) The depletion of mBet3p by RNAi disrupts the architecture of the Golgi. siRNA targeted to mBet3p (BetC, right) or luciferase as a control (left) were transfected into COS-7 cells. siRNA-transfected cells were stained with antibodies directed against mBet3p (top) and GM130 (bottom). (D) β-tubulin was unaffected in cells transfected with mBet3-specific siRNA. Cells transfected with siRNA targeted to luciferase (left) and mBet3p (right) were stained with α-mBet3p (top) and anti-β-tubulin antibody (bottom). Error bars represent the SEM. Bars: (A) 200 nm; (C and D) 10 µm.