Extended Data Fig. 10: Analysis of the functional interaction between AMFR and FAM134B in cells and in vitro.
From: Ubiquitination regulates ER-phagy and remodelling of endoplasmic reticulum
a, HeLa cells were transfected with control siRNA (siNT), siRNA#1 or siRNA#2 targeting AMFR (siAMFR) for 72 h. Detergent-soluble protein extracts were analysed by western blot using antibodies against FAM134B, AMFR or vinculin (loading control). b, Densitometric quantification of endogenous FAM134B (normalised to vinculin) in panel a (Data are means ± s.d. of n = 5 independent experiments, One-way ANOVA, Bonferroni post-hoc test). c, Confocal fluorescence microscopy of U2OS cells stably expressing HA-FAM134BWT transfected with either control siRNA (siNT) or siRNA#1 targeting AMFR (siAMFR) for 72 h, followed by incubation with 250 nM Torin 1 for 6 h. Cells were fixed and stained for HA-FAM134B and endogenous LC3B, respectively. d, Quantification of HA-FAM134B-WT/LC3B-II-containing puncta per cell of images in panel c. Scatter plot graphs represent means ± s.d. (nsiNT = 24 cells, nsiAMFR = 33 cells; two-tailed Mann-Whitney-U-test). e, HeLa cells were treated with 250 nM Torin 1 for the indicated time (h). Densitometric quantification of endogenous AMFR (normalised to vinculin), as presented in Fig. 5a. Data are means ± s.d. of n = 3 independent experiments; two-way ANOVA, Bonferroni post-hoc test. f, U2OS cells stably expressing HA-FAM134BWT or HA-FAM134B-17KR were incubated with 250 nM Torin 1 for 0, 2, 4, 6 and 8 h. Detergent-soluble extracts were analysed by western blot using antibodies against HA, AMFR and vinculin. g,h, Densitometric quantification of HA-FAM134B and AMFR (normalised to vinculin) from panel f. Data are means ± s.d. of n = 3 independent experiments, two-way ANOVA, Bonferroni post-hoc test. i, U2OS cells stably expressing HA-FAM134BWT or HA-FAM134B-LIR incubated with 250 nM Torin 1 for 0, 2, 4, 6 and 8 h. Detergent-soluble extracts were analysed by western blot using antibodies against HA, AMFR and vinculin. j, Densitometric quantification of AMFR (normalised to vinculin) in panel i. Data are means ± s.d. of n = 3 independent experiments, two-way ANOVA, Bonferroni post-hoc test. k, and l, Purification of AMFR from HEK 293T cells. m,n, Mass spectrometry (MS) analysis of the in vitro ubiquitination of full length GST-tagged FAM134B using recombinant AMFR. (Data are means ± s.d. of n = 3 independent experiments; two-tailed unpaired Student’s t-test). o–q, MS analysis of the in vitro ubiquitination of His-RHD90–264-Strept-II and His-Ub-RHD90–264-Ub-Strept-II using recombinant AMFR. Data are means ± s.d. of n = 3 independent experiments; two-tailed unpaired Student’s t-test. r, Immunodetection of native His-RHD90–264-Strept-II following ubiquitination by AMFR. The ubiquitination reaction was analysed by western blot after blue native polyacrylamide gel electrophoresis (BN-PAGE) using antibodies against His6 or UbP4D1. Control reaction (in the presence of AMFR, no ATP).
