Fig. 2

UVRAG^FS prevents TLR4 induction of autophagy in vivo. a Representative images (left panels) and quantification (right panels) of GFP-LC3 puncta in indicated tissues from Dox-treated/untreated iUVRAG^FS mice and littermate control mice crossed with GFP-LC3 transgenic mice, challenged with PBS or LPS (20 mg per kg body weight; i.p.). Arrows denote GFP-LC3 puncta. Scale bars, 10 μm. n = 10 tissue sections pooled from three independent experiments. b UVRAG^FS inhibits the Beclin1-autophagy interactome and K63-linked ubiquitination in LPS-stimulated bone marrow-derived macrophages (BMDMs). BMDMs derived from iUVRAG^FS mice were incubated with PBS or LPS (100 ng/ml) for 4 h in the presence/absence of Dox and subjected to IP with anti-Beclin1 followed by WB of indicated proteins (left panel). Expression of indicated proteins in WCL were shown (middle panel). Densitometric quantification of the Bcl-2-associated Beclin1/total Beclin1 and the ubiquitinated (K63)-Beclin1/total Beclin1 ratios in BMDM are also shown (right panels). c UVRAG^FS prevents LPS-induced PI3KC3 activation in BMDM. Cells cultured as in b were transfected with p40(phox)-PX-GFP and subjected to confocal microscopy. Representative images of p40(phox)-PX-GFP puncta in indicated BMDM are shown (left) and the number of p40(phox)-PX-GFP puncta per cell was quantified (right). Scale bars, 10 μm. n = 10–15 high-power fields (HPF) pooled from three independent experiments. Data in b are from one experiment that is representative of four independent experiments. For all quantifications, data (mean ± SD) were from the indicated number of independent experiments and analyzed with two-way ANOVA. n.s., not significant; ****P < 0.0001. Source data are provided as a Source Data file. See Supplementary Fig. 9 for uncropped data of b.