Fig. 4: Ubiquitylation of DRP1 by TRIM25.

A Cell transfected with the indicated GFP-tagged constructs were lysed, immunoprecipitated and immunoblotted. Note differential ISGylation profiles of DRP1 amongst the samples. Reverse co-IP confirms the same result. The input levels of DRP1, ISG15 and VINCULIN in the total lysates served as loading controls. B Cells transfected as in panel A were immunoprecipitated (IP) with anti-DRP1 antibody. Western blot (IB) analysis against HA-tagged Ub reveals increased DRP1 ubiquitylation in CoV2 PLpro WT samples. DRP1 and VINCULIN levels in the total lysates served as loading controls. C Mock or HERC5 siRNAs-treated cell lysates were immunoprecipitated (IP) with anti-DRP1 antibody. Ex vivo ubiquitylation of DRP1 was analysed by immunoblotting against Ub. Lysates were also checked for the levels of DRP1, HERC5 and VINCULIN. D Cells transfected with indicated GFP-tagged constructs were treated MG132 or left untreated. Note increased DRP1 levels upon drug treatment. Immunoblots were analysed against DRP1 and VINCULIN. Graph shows changes in protein levels of DRP1. Data represents 3 independent experiments. ns, not significant (p > 0.07), *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001 using unpaired 2-tailed Student’s t-test. E Cells were transfected with indicated mCherry-tagged constructs, immunoprecipitated against DRP1 and immunoblotted with Ub antibody. Note that K532R mutation in DRP1 compromises its ubiquitylation. endogenous DRP1, ← mCherry-tagged DRP1. VINCULIN was used as loading control. F Cells transfected with CoV2 PLpro WT were treated with the indicated siRNAs and immunoblotted against DRP1. Knockdown efficiency was confirmed by immunoblotting against PARKIN, TRIM25 and MITOL. VINCULIN was used as loading control. Graph shows the changes in the protein levels of DRP1. Data represents 3 independent experiments. ns, not significant (p > 0.07), ***p ≤ 0.001 using unpaired 2-tailed Student’s t-test. G Mock or TRIM25 siRNAs treated cells transfected with indicated GFP-tagged constructs were immunoblotted with anti-DRP1 antibody. Note increased DRP1 levels upon TRIM25 depletion. Input levels of TRIM25 and VINCULIN served as loading controls. Graph plots protein levels of DRP1. Data represents 3 independent experiments. ns, not significant (p > 0.08), *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001 using unpaired 2-tailed Student’s t-test. H A549 cell lysates were immunoprecipitated against DRP1 and immunoblotted with TRIM25 antibody. The proportion of lysate loaded as input and used for immunoprecipitation is denoted in brackets by ‘X’. I Mock or TRIM25 siRNAs treated cells were co-immunoprecipitated against ISG15 and DRP1. Note similar ISGylated DRP1 levels across samples. TRIM25 levels in the lysates confirm knockdown efficiency, VINCULIN served as loading control. DRP1 levels were also checked in cell lysates. J Cells expressing the indicated GFP-tagged constructs were depleted off TRIM25 and analysed for co-IP between DRP1 and Ub. Note significantly reduced DRP1 ubiquitylation in TRIM25 depleted cells expressing CoV2 PLpro WT. Lysates were immunoblotted against DRP1, TRIM25 and VINCULIN.