Fig. 4.

ATP13A1 depletion has no visible effect on Bag6-MAVS interaction. (A) Proposed model. Bag6 recruits MAVS after its ATP13A1-mediated extraction from the ER membrane. Depleting ATP13A1 will decrease Bag6-MAVS interaction, as MAVS cannot be dislocated from the ER membrane. (B) ATP13A1 depletion does not grossly alter the levels of MAVS in the crude cytosolic supernatant fraction. (i) Control KO cells transfected with non-targeting (nt) or ATP13A1-targeting siRNAs (siATP13A1) were fractionated as shown in Fig. 2A. Equivalent amounts of supernatant (S) and pellet (P) fractions were analysed by immunoblotting for the indicated endogenous proteins. (ii) ATP13A1 and MAVS levels in siATP13A1-treated cells relative to nt siRNA-treated cells, where protein levels were set to 1. Shown are means±s.e.m. for five biological replicates as shown in Bi. ****P<0.0001; ns, not significant (two-tailed one-sample t-test). (iii) Mean±s.e.m. of the supernatant/total ratio of MAVS levels in siATP13A1-treated cells relative to the respective ratio in nt siRNA-treated cells for five independent experiments as in Bi. Same colour data points correspond to a single biological replicate; ns, not significant (paired two-tailed t-test). (C) ATP13A1 depletion does not affect Bag6-MAVS interaction. Supernatant (S) fractions from Bi were subjected to immunoprecipitations with rabbit anti-Bag6 antibody (αBag6) or rabbit control IgG antibody. Inputs and immunoprecipitates were analysed by immunoblotting for the indicated endogenous proteins. SGTA served as loading control as well as an internal control for equal Bag6 co-immunoprecipitation potential. Arrow in Bi and C indicates full-length MAVS. Open circles on MAVS blots indicate signals derived from denatured antibody heavy and light chains. (D) Mean±s.e.m. of MAVS levels that co-immunoprecipitate with Bag6 in siATP13A1-treated cells relative to nt siRNA-treated cells for four independent experiments as shown in C. ns, not significant (paired two-tailed t-test).