Research Paper Volume 12, Issue 24 pp 25564—25580

Figure 2. Identification of the binding motifs involved in TRIM27-SIX3 interaction. Schematic representation of FLAG-tagged full-length (A) TRIM27 or (C) SIX3 (FL), along with its various deletion mutants (D1 and D2). CC, coiled-coil domain. A549 cells were cotransfected with the (B) indicated HA-tagged SIX3 constructs along with those encoding FLAG-tagged TRIM27 or the (D) indicated FLAG-tagged TRIM27 constructs along with those encoding HA-tagged SIX3. Interaction between TRIM27 and SIX3 was determined by immunoprecipitation and immunoblotting. (E–G) A549 cells infected with pLVX-Puro-TRIM27 or blank pLVX-Puro vector were treated with DMSO or 10 μM MG132 for 4 h, and SIX3 expression was determined by quantitative real-time PCR and western blot analysis. (H, I) A549 cells infected with pLVX-Puro-TRIM27 or blank pLVX-Puro vector were treated with CHX (100 μg/mL), and SIX3 expression was determined by western blot analysis. (J) SIX3 was immunoprecipitated and immunoblotted in A549 cells transfected with siTRIM27-1 or siNC. (K) A549 cells were cotransfected with the SIX3 (WT) or mutant SIX3 constructs along with myc-TRIM27 and His-Ub constructs, and a pull-down assay was performed. All experiments were repeated at least three times, and data are represented as mean ± SD. (F) ***P < 0.001 (two-way ANOVA followed by Dunnett’s test).