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. 2017 Jul 25;13(7):e1006932. doi: 10.1371/journal.pgen.1006932

Fig 2. MLF and DnaJ-1 bind Lz and control its stability and activity.

Fig 2

(A) Luciferase assays in Kc167 cells treated with the indicated dsRNA and transfected with 4xPPO2-Fluc reporter plasmid in the presence or not (ctr) of the pAc-Lz-V5 expression plasmid. pAc-Rluc was used as an internal normalization control. (B) Western blots showing Lz-V5, MLF, Renilla luciferase (R luc) and Tubulin (Tub) expression in Kc167 cells treated with the indicated dsRNA and cotransfected with pAc-Lz-V5 and pAc-Rluc expression vectors. (A, B) dsDnaJ-1 (a) and (b) correspond to two distinct dsRNAs targeting dnaj-1. Of note, the multiple bands for Lz are only observed using C terminally (V5) tagged versions of Lz and not with N terminally (GFP) tagged Lz; they likely represent internal translation initiation events. The multiple bands observed using a MLF antibody could represent different MLF protein isoforms as described in [17]. (C, D) Luciferase assays (C) and Western blots (D) performed on Kc167 cells transfected with the 4xPPO2-Fluc reporter plasmid and pAc-based expression plasmids for Lz and for different DnaJ-1 variants as indicated. Rluc and Tubulin were used as internal controls. (E, F) Western blots showing the results of immunoprecipitation experiments against GFP performed in Kc167 cells transfected with expression vectors for HA-MLF (E) or HA-DnaJ-1 (F) and GFP or GFP-Lz as indicated in the upper part of the panels. (G, H) Western blots showing the results of immunoprecipitation experiments against GFP performed in Kc167 cells transfected with expression vectors for GFP-Lz and various HA-MLF (G) or HA-DnaJ-1 (H) mutants. (A, C) For luciferase assays means and standard deviations of results from biological triplicates are shown. ***: p-value<0.001, **: p-value<0.01 (Students t-tests) as compared to Lz with dsGFP condition.