Extended Data Fig. 7: Vacuole-mediated clearance of INQ and JUNQ.
Representative images of WT cells expressing NES-2xKeima-LuciTs after 2 hr incubation at 37 °C with 100μM MG132. Over time, fluorescence is seen with excitation in the 558 nm channel indicating the NLS-LuciTs has encountered an acidic environment. Insets show the transition from green to red and a structure leaving the inclusion that is fully red. Scale bars on large images are 5 mm. Scale bars on magnifications are 1 μm. Same data shown in Fig. 7d, but with more time points and a larger field of view in the images. Scale bars on large images are 5 μm. Scale bars on magnifications are 1 μm. (b) WT cells expressing NES-2xKeima-LuciTs after 85 min incubation at 37 °C with 100μM MG132. (c) Longer exposure of the blot shown in Fig. 7f to highlight the difference in the number and pattern of the EGFP bands in the WT vs pep4Δ cells. (d) Levels of EGFP at time 0 were measured from Quantitative Western blots such as those shown in Fig. 7e, f (mean ± S.E.M. from three biologically independent experiments). WT and pep4Δ yeast were compared using a two-tailed paired Student’s t-test without reaching statistical significance. (e) WT yeast expressing NLS-EGFP-LuciTs were treated with 8μM of FM4-64 and incubated for 2hr at 37 °C with 100μM MG132. Cells were imaged every 30 sec for 90 mins. Scale bar is 1μm. Same data shown in Fig. 7h, but only WT and with more timepoints during the entry into the vacuole. Source numerical data and unprocessed blots are available in source data.
