Fig. 2. Mint3 is required for the localization of Furin to the TGN. (A) Knockdown of endogenous Mint3. HeLa cells were transiently transfected with either empty vector (control), non-target control siRNA (NT) or human Mint3 shRNA targeting plasmids (RNAi-1 and RNAi-2). Equal amounts of total protein were analyzed by immunoblot as shown by detected levels of β-actin. The quantification of the immunoblots is shown in the graph. Each value represents the mean ± s.e.m. of triplicate experiments. Asterisks denote significant differences from control cells (P<0.05), as determined by Student's t-test. (B) Loss of Mint3 significantly disrupts the localization of Furin to the TGN. Arrowheads indicate cells in which Mint3 expression was suppressed by RNAi. Bar graphs shows the proportion of cells with Mint3 knockdown (out of 100 cells counted on each of two different slides for each condition). Asterisks denote significant differences from control cells (P<0.05), as determined by Student's t-test. (C) Subcellular localization of Furin in control and Mint3-knockdown cells. HeLa cells transiently transfected with either empty vector (control) or human Mint3 shRNA plasmid (RNAi-2) were fixed and immunostained with antibodies to Furin and either TGN46, KDEL or EEA1 as indicated. The enlarged regions of the boxed areas are shown to the right. Scale bars: 5 µm.