Figure 4
From: Mice lacking WRB reveal differential biogenesis requirements of tail-anchored proteins in vivo
The cytoplasmic domain of Syntaxin 5 is aggregation-prone and can induce autophagy.
(a) Stx5op and Stx8op were translated in vitro in a rabbit reticulocyte lysate or in a lysate immunodepleted of TRC40. (b) Integration into rough microsomal membranes (RM) was detected by glycosylation of the C-terminal opsin tag (opG) resulting in a slower-migrating protein detected by an opsin-specific antibody. (c) Quantification of glycosylation, percentage of total. Bars represent average +/−s.e.m. (n = 4, ***p-value < 0.001). (d) Stably transfected HEK Flp-In T-REx cells were cultured in absence or presence of tetracycline for 16 hours to induce expression of Stx5/Stx8 constructs. Cellular lysates were separated by SDS-PAGE and induction was verified by anti-GFP western blot. An increased ratio between the lipidated (LC3B-II) compared to non-lipidated (LC3B-I) LC3B was evaluated by blot and quantified in e Bars represent average +/−s.e.m. (n = 4, *p-value < 0.05; ***p-value < 0.001). (f) Far-UV circular dichroism measured at 220 nm and increasing temperatures for MBP-Stx5cyt (black trace) and MBP-Stx8cyt (gray trace).
