Figure 5: TBC1d5 is required for retromer-mediated trafficking but not endosomal recruitment of VPS35.
From: Structural and mechanistic insights into regulation of the retromer coat by TBC1d5
(a) Levels of TBC1d5, VPS35 and actin expression in TBC1d5 KO (clone 1 and 2) HeLa cell line and parental cells determined by immunoblotting. (b) Parental and clone 1 HeLa cells were mixed with 1:1, fixed and stained for TBC1d5 (red) and VPS35 (green). Asterisk represents cell that is devoid of TBC1d5. Scale bar, 10 μm. (c) Parental and clone 1 HeLa cells were mixed with 1:1 and incubated with anti-CD49e antibody. The cells were then fixed and stained for TBC1d5 (red), VPS35 (green) and CD49e (magenta). Asterisk represents cell that is devoid of TBC1d5. Scale bar, 10 μm. (d) The average mean fluorescence intensity (MFI) of retromer-localized CD49e in parental, clone 1 or clone 2 HeLa cells, with error bar representing s.e.m. Cells were fed anti-CD49e antibody for 1 h, then fixed and stained for internalized CD49e and VPS35. MFI was measured using Zen2009 software. One hundred cells from each group were used for comparison. (e) TBC1d5 is important for CI-MPR dispersal. Parental, clone 1 or clone 2 Hela cells were fixed and stained with anti-CI-MPR. Scale bar, 20 μm. (f) Quantification of e. One hundred cells from each group were scored for either compact or dispersed CI-MPR distributions.
