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. Author manuscript; available in PMC: 2021 Feb 3.
Published in final edited form as: Nat Cell Biol. 2020 Aug 3;22(8):973–985. doi: 10.1038/s41556-020-0549-1

Extended Data Fig. 3. IRGM effects on mTOR and calcineurin and mAtg8s interactions with and effects on TFEB.

Extended Data Fig. 3

a, b, Western blot analysis and quantifications of the effects of IRGM KD on pTFEB (S211) levels in cells treated with pp242. Data, means ± SEM of normalized intensities (n=3 biologically independent experiments) ANOVA, Tukey’s post hoc test. c–e, western blots analysis of the effects of IRGM on mTOR substrates pS6K and pULK1. Data, means ± SEM of normalized intensities (n=3 biologically independent experiments) paired t-test. f, HCM image analysis of co-localization between mTOR and LAMP2. Scale bar 10 μm. g,h, HCM analysis of the effects of IRGM KD on LAMP2 puncta. Data, means ± SEM; (n=3 biologically independent experiments) paired t-test. Scale bar 10 μm. i, HCM analysis of the effect of IRGM expression on cells expressing RagBQ99L and parental 293T cells on nuclear translocation of TFEB, (n=3 biologically independent experiments). Scale bar 10 μm. j, confocal microscopy analysis of co-localization between GFP-IRGM and endogenous PPP3CB in HeLa cells (n=3 biologically independent experiments). Scale bar 5 μm. k,l, HCM analysis of the effect of starvation on colocalization between GFP-IRGM and PPP3CB. Data, means ± SEM (n=3 biologically independent experiments) paired t-test. Scale bar 10 μm. m, western blot showing PPP3CB KD in HeLa cells (n=3 biologically independent experiments). n, schematics of LysoIP technique. o, LysoIP to detect indicated proteins on lysosomes (n=3 biologically independent experiments). p, western blot analysis of the effects of IRGM expression on NFAT mobility shift (n=3 biologically independent experiments). q, Co-IP analysis of GFP-LC3B and GFP-GABARAP with FLAG-TFEB in 293T cells. r, GST pull-down analysis of TFEB with WT or LDS mutant of GABARAP. s, HCM images in WT or HexaKO cells (n=3 biologically independent experiments). Scale bar 10 μm. Uncropped blots for panels a, c, o, p, q and r are provided in Unprocessed Blots Data Extended Fig. 3 and numerical source data for panels b, d, e, g and l are provided in Statistical Source Data Extended Fig. 3.