Fig. 6: EFHD2 interacts with complex Ι and inhibits the internalization of TNFR1. | Nature Communications

Fig. 6: EFHD2 interacts with complex Ι and inhibits the internalization of TNFR1.

From: EFHD2 suppresses intestinal inflammation by blocking intestinal epithelial cell TNFR1 internalization and cell death

Fig. 6

a Lysates from HCT-116 cells without or with overexpression of Flag-EFHD2 treated with 50 ng/ml TNF for the indicated time were immunoprecipitated with Flag M2 beads followed by western blot analysis for indicated molecules of TNFR1-complex Ι. Quantifications of the indicated proteins in TNFR1-complex Ι were shown below. b Lysates from HCT-116 cells treated with TNF for indicated time were immunoprecipitated with RIPK1 to analyze EFHD2 interaction by western blot. The quantification of EFHD2/RIPK1 ratio was shown below. Representative flow cytometric analysis plot (c) and proportions (d) of cell-surface TNFR1-positive cells from WT and EFHD2-/- HT-29 cells treated with TC, or TS for the indicated time. e Representative immunofluorescence detection of TNFR1 (red) and EEA1 (green) from WT and EFHD2-/- HT-29 cells treated with TS for 30 min. Scale bar, 10 μm. f Western blot detection of lysates from isolated early endosome from WT and EFHD2-/- HT-29 cells treated with TS for 30 min (left) and quantifications of TNFR1/EEA1 and TNFR1/Rab5 ratio in early endosome by ImageJ (right). g Flow cytometric analysis of caspase-3/7 activity from dispersed WT and Efhd2-/- enteroids treated with 20 ng/ml TNF for 24 hours, without or with MDC pretreatment for 1 hour. n = 3 (a, b, d, f, g). Data are representative of three independent experiments; error bars show means ± s.d. P values were determined by unpaired two-tailed t-test in b, d, f, g. Two-way ANOVA analysis with Sidak’s multiple comparisons test in a. Source data are provided as a Source Data file.

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