Extended Data Fig. 6: SHISA9 bridges IKKi to autophagy membrane protein GABARAPL1.
From: Modulation of virus-induced neuroinflammation by the autophagy receptor SHISA9 in mice
(a) Immunoblotting analysis of extracts of wild-type (WT), SQSTM1 knockout (KO), OPTN KO or CALCOCO2 KO HEK293T cells transfected with empty vector or increasing amounts of Myc-SHISA9, with EBSS treatment. (b) Immunoblotting analysis of extracts of U87 cells treated with DMSO or 3-MA (10 mM). (c) Co-immunoprecipitation (IP) and immunoblotting analysis of extracts of HEK293T cells transfected with HA-SHISA9 and indicated Flag tagged ATG8 family members. (d) Co-immunoprecipitation and immunoblotting analysis of extracts of Flag-SHISA9-inducible cells treated with or without doxycycline (Dox, 100 ng/ml) for 12 hrs, followed by immunoprecipitation with anti-Flag. (e) Purified Flag-GABARAP1/ GABARAPL1 and His-tagged SHISA9 were incubated together, followed by immunoprecipitation with anti-His and immunoblotting analysis. (f) Domain organization of SHISA9 protein. (g) Co-immunoprecipitation and immunoblotting analysis of extracts of U87 cells transfected with Flag-GABARAPL1, and Myc-SHISA9 or its indicated mutations, followed by HSV-1 (MOI = 1) infection for 24 hrs. (h) Immunoblotting analysis of extracts of U87 cells transfected with WT Flag-SHISA9 or its F170A mutant, with or without HSV-1 (MOI = 1) infection for 24 hrs. (h) Co-immunoprecipitation and immunoblotting analysis of extracts of U87 cells transfected with HA-GABARAPL1, Flag-IKKi and Myc-SHISA9 with indicated combinations, followed by HSV-1(MOI = 1) infection for 24 hrs. (j) Co-immunoprecipitation and immunoblotting analysis of extracts of HEK293T cells transfected with Flag-SHISA9 (WT, F170A) and HA-IKKi. (k) Immunoblotting analysis of extracts of U87 cells transfected with Flag-SHISA9 and its indicated mutations. (l) Quantification of the indicated proteins levels of Fig. 5k by using ImageJ software. P-values were determined by unpaired two-tailed Student’s t test. For (a-c, j and k), EBSS treatment were for 6 hrs. For (c, g, h and j), chloroquine (CQ, 50 μM) was added in all groups for 6 hrs. For (a-e, g-k), similar results were obtained by three independent biological experiments.
