Extended Data Figure 5: Further in vivo and in vitro analysis of mutant phenotypes associated with Get2_TM2-1sc.

a, GFP expression of the heat-shock reporter in the indicated strains was measured by flow cytometry analysis as in Fig. 1a (n = 2). b, Representative confocal microscopy images of Sgt2–mCherry localization in the indicated strains. White arrowheads indicate the presence of cytosolic tail-anchored protein aggregates. c, Equal amounts of microsomes (normalized by D_280 nm) were subjected to SDS–PAGE analysis and visualized by immunoblotting. d, The indicated microsomes were solubilized in Triton X-100 detergent or mock-solubilized on ice for 30 min. TEV protease digestion of samples, as indicated, was performed overnight at 4 °C followed by SDS–PAGE analysis and immunoblotting. e, Affinity-purified Get3–Sec22 was incubated with the indicated microsomes and analysed by Sec22 insertion analysis as in Fig. 1b. f, Affinity-purified Get3–Sec22 was incubated with the indicated microsomes or mock-incubated and analysed by crosslinking analysis as in Fig. 1c.