Extended Data Fig. 2: ADSL T350 phosphorylation drives ER translocation and ADSL-STING association via KDELR3 Interaction.
From: ADSL-generated fumarate binds and inhibits STING to promote tumour immune evasion
Immunoprecipitation and immunoblotting assays were performed with the indicated antibodies, representative results from three independent experiments are shown. For immunofluorescence analyses, the area outlined in white is magnified and is shown on the right; 50 cells from three independent experiments were analysed and representative data are shown. (a) Alignment of protein sequences spanning ADSL ER translocation signal from different species. (b) Bacterially purified His-ADSL WT, E481/L482A, T350A and A291V were used for ADSL activity detection (n = 3 biological experiments). (c) BT-549 cells transfected with Flag-ADSL WT or E481/L482A were treated with or without hypoxia. ER fractions and total lysates were harvested for immunoblotting analyses as indicated. (d) BT-549 cells transfected with the indicated plasmids were treated with or without hypoxia. (e) Bacterially purified GST-ADSL WT, T350A or E481/L482A protein immobilized on glutathione agarose beads was mixed with or without active His-IKKβ for an in vitro kinase assay. The protein were then incubated with His-KDELR3 protein. A GST pulldown assay was performed, and the precipitated protein were incubated with or without CIP (10 U) for 30 min at 37 °C. (f) Parental BT-549 cells and the indicated clones with knock-in expression of ADSL T350A, and E481/L482A were treated with or without hypoxia for 6 h (upper). The colocalization coefficients between ADSL and calnexin are shown (lower). (g) Parental BT-549 and MDA-MB-231 cells, as well as the corresponding clones expressing knock-in ADSL A291V, were treated with or without hypoxia. (h) Parental BT-549 cells and the indicated clones with knock-in expression of ADSL A291V were treated with hypoxia for 6 h (upper). The colocalization coefficients between ADSL and calnexin are shown (lower). (i-k) BT-549 cells with and without KDELR3 shRNA expression were treated with hypoxia for 6 h (i, upper). The colocalization coefficients between the indicated protein are shown (i, lower). ER fractions and total lysates were harvested for immunoblotting analyses (j). Total lysates were harvested for immunoprecipitation and immunoblotting analyses (k). Data are representative of as mean ± SD; Statistical significance was determined by two-tailed Student’s t-test.
