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. 2022 May 11;135(9):jcs259596. doi: 10.1242/jcs.259596

Fig. 5.

Fig. 5.

EMC5 depletion enhances Bag6–MAVS interaction. (A) Proposed model. Bag6 binds MAVS before its EMC-mediated integration at the ER membrane. EMC deficiency will promote Bag6 binding to the cytosolic pool of MAVS that fails to be imported into the ER membrane. (B) EMC5 depletion does not grossly alter the levels of MAVS in the crude cytosolic supernatant fraction. (i,ii) Control KO cells transfected with non-targeting (nt) or EMC5-targeting siRNAs (siEMC5) 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. Blots resulting from the same membrane are clustered together. Tubulin and OST48 serve as loading controls for the supernatant and pellet fractions, respectively. (iii) EMC subunit and MAVS levels in siEMC5-treated cells relative to nt siRNA-treated cells, where protein levels were set to 1. Shown are means±s.e.m. for three-five biological replicates as shown in Bi,ii. ****P<0.0001; **P<0.01; ns, not significant (one-way ANOVA with Tukey's multiple comparison tests). (iv) Mean±s.e.m. of the supernatant/total ratio of MAVS levels in siEMC5-treated cells relative to the respective ratio in nt siRNA-treated cells for five independent experiments as in Bii. Same colour data points correspond to a single biological replicate; ns, not significant (paired two-tailed t-test). (C) EMC5 depletion enhances Bag6-MAVS interaction. Supernatant (S) fractions from Bi,ii 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. In Bi, the EMC2 and EMC1 bands are indicated by arrows. Arrow in Bii 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 siEMC5-treated cells relative to nt siRNA-treated cells for four independent experiments as shown in C. *P<0.05 (paired two-tailed t-test).