Figure 6.

Poly I:C–induced TRIM28 phosphorylation hinders the formation of aggresomes containing GFP-CFTR-ΔF508. HeLa cells stably expressing GFP-CFTR-ΔF508 were treated with a control siRNA or TRIM28 siRNA. One day later, the cells were transfected with a plasmid expressing siRNA-resistant FLAG-TRIM28[R] (WT, S473A, or S473E) or a plasmid expressing only FLAG, which served as a negative control. One day later, the cells were either mock-transfected or transfected with poly I:C. The cells were treated with MG132 for 12 h before cell fixation; n = 3. (A) Western blotting validating specific downregulation of endogenous TRIM28 by means of siRNA and the expression of FLAG-TRIM28[R]-WT, FLAG-TRIM28[R]^S473A, or FLAG-TRIM28[R]^S473E at a level comparable to that of endogenous TRIM28. (B) Immunostaining of GFP-CFTR-ΔF508 (green) and exogenously expressed FLAG-TRIM28[R]-WT, FLAG-TRIM28[R]^S473A, or FLAG-TRIM28[R]^S473E (red). Nuclei were stained with DAPI (blue). Scale bar: 10 μm. (C) Relative percentages of cells containing either aggresomal or dispersed GFP-CFTR-ΔF508. To accurately assess the effect of exogenously expressed FLAG-TRIM28, only the cells expressing both GFP-CFTR-ΔF508 and exogenous FLAG-TRIM28 were counted. Two-tailed, equal-sample variance Student’s t test was carried out to calculate the P values. *, P < 0.05; **, P < 0.01