Fig. 6: NOC4L directly interacted with TLR4.
From: Macrophage deletion of Noc4l triggers endosomal TLR4/TRIF signal and leads to insulin resistance
a HeLa cells were co-transfected with NOC4L plasmids with 3×FLAG tag and EGFP–TLR4 plasmids, and cell lysates were prepared for co-immunoprecipitation. Cell lysates (Input) and FLAG immunoprecipitation were determined by immunoblot analysis for FLAG and GFP. b Endogenous Noc4l and TLR4 interaction in RAW264.7 cells. Cell lysates (Input) and Noc4l (6 R) immunoprecipitation analyzed by immunoblot analysis for TLR4 and Noc4l. c Endogenous Noc4l and TLR4 interaction in BMDMs isolated from Noc4lfl/fl and Noc4lLKO mice. Cell lysates (Input) and Noc4l (6 R) immunoprecipitation analyzed by immunoblot analysis for TLR4 and Noc4l. d Endogenous Noc4l and TLR4 interaction in RAW264.7 cells stimulated with LPS (200 ng/mL). Cell lysates (Input) and Noc4l (6 R) immunoprecipitation analyzed by immunoblot analysis for TLR4 and Noc4l. e Representative images of Proximity Ligation Analysis (PLA). PMs of Noc4lfl/fl, Noc4lLKO, and Noc4lLOE mice were stained with the rabbit anti-TLR4 antibody and mouse anti-Noc4l antibody (3L7) or IgG antibody, and in vivo protein–protein interaction between TLR4 and Noc4l (denoted by a red dot) was detected with secondary proximity probes, Anti-Rabbit MINUS and Anti-Mouse PLUS, using the Duolink protein–protein interaction detection kit in vivo. f Quantification of positive signals in each cell of each group in e. Two-tailed Student’s t test. ****p < 0.0001. g PLA was performed in PMs of WT and TLR4 mutant mice as described in e. h Indicated truncates of TLR4 were constructed according to their functional domains. Full-length Flag-NOC4L-His and GST-TLR4 were expressed and purified from Sf9 cells and BL21 respectively, and then GST pulldown assays were performed. Purified GST-TLR4 proteins and Flag-NOC4L-His proteins were incubated with GST beads overnight at 4 °C. NOC4L was analyzed by Western blotting using anti-Flag antibody. GST-TLR4-1: 1–199aa, GST-TLR4-2: 200–423aa, GST-TLR4-3: 424–673aa, GST-TLR4-4: 674–839aa. Data represented three independent experiments. All data are presented as mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001. Source data are provided as a Source Data file.
