Fig. 4: Hmx1 is extracted from class E compartments for EGAD.
a SDS–PAGE and WB analysis with the indicated antibodies. NG-ALFA-Hmx1 (induced with 500 nM ß-estradiol (ß-ES) for 90 min) in vps4Δ and tul1Δ vps4Δ mutant cells. Cells were left untreated (0 min) or treated with CHX for the indicated time to block protein synthesis. Densitometric quantification is shown in Supplementary Fig. 4a. b Live cell epifluorescence microscopy: NG-ALFA-Hmx1 (green) and mCherry-Cps1 (red). Class E compartments (E) are indicated; scale bars 5 µm, **** p < 0.0001. Data were analyzed by a two-tailed unpaired t-test. Quantification of mean fluorescence intensity of NG-Hmx1 on class E compartments before and after CHX addition (n = 10 individual class E compartments). The mean fluorescence intensity of the individual class E compartments is presented as box plots, with lower and upper quartile and median, and with min. and max. values as whiskers. c SDS–PAGE and Western blot analysis with the indicated antibodies (including a K48 linkage specific antibody) of denaturing FLAG-Hmx1 immunoprecipitations (IP) from the indicated cells. d, h SDS–PAGE and Western blot analysis with the indicated antibodies of denaturing FLAG-Hmx1 immunoprecipitations (IP) from d vps4Δ cells with CDC48 or cdc48-3 mutants (in a pdr5Δ vps4Δ background) or h vps4Δ cells expressing Ubx3ΔUBX, left untreated or treated with 50 µM MG-132. S100 and P100 fractions were subjected to denaturing FLAG-Hmx1 immunoprecipitations (IP). e Densitometric quantification of ubiquitinated FLAG-Hmx1 species from S100 fractions (n = 3 independent experiments, presented as mean ± standard error of the mean (SEM)) from Fig. 4d. Data were analyzed by a two-tailed unpaired t-test. f, g SDS–PAGE and Western blot analysis of total cell extracts from the indicated cells. f Cells were left untreated or treated with 50 µg/mL CHX for 90 min and 180 min. Pgk1 served as a loading control. g NG-Hmx1 expression was induced with 500 nM ß-estradiol (ß-ES) for 90 min prior to the addition of 50 µg/mL cycloheximide (CHX). Pgk1 served as a loading control. The densitometric quantification of (f, g) is shown in Supplementary Fig. 4d, e. For Orm2 see Supplementary Fig. 4f, g. Representative blots from 3 independent experiments with similar results are shown in c, f, g, h.
