Figure 4.

The S473 residue is responsible for TRIM28-mediated inhibition of aggresome formation. (A) A schematic diagram of the TRIM28 protein. The mutation sites are indicated by solid circles. Deletion variants of TRIM28 with the N-terminal FLAG-tag are also indicated. R, RING domain; BB, two B-box-type zinc finger domains; CC, coiled-coil motif; H, HP1-binding domain; P, PHD domain; and B, bromodomain. (B) Immunostaining of GFP-CFTR-ΔF508 and FLAG-TRIM28, either WT or its variant. HeLa cells stably expressing GFP-CFTR-ΔF508 were transiently transfected with a plasmid expressing FLAG, FLAG-TRIM28-WT, or its variant. Two days later, the cells were treated with MG132 for 12 h before fixation. The cells were stained with the anti-GFP antibody (green) and an anti-FLAG antibody (red). Nuclei were stained with DAPI (blue). Scale bar: 10 μm; n = 3. (C) Relative distribution of GFP-CFTR-ΔF508. Relative percentages were determined by counting the cells containing either the aggresome or the dispersed aggregates of GFP-CFTR-ΔF508 in the immunostaining images. 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 < 0.01