Figure 5.

RPP DLC-AS enhances nuclear localization mediated by T-agNLS dependent on MT integrity and LC8 association. Cells were transfected with the indicated GFP fusion protein and CLSM images were used to calculate the Fn/c ratio as described in the legend to Figure 2. (A) CLSM images of representative cells are shown, with the brightness of all images increased using Image J software. Cytoplasmic fluorescence can be discerned in GFP-T-agNLS with and without NCZ treatment, but little or no cytoplasmic fluorescence can be seen in cells expressing GFP-RPP_139-174-T-agNLS unless the cells are treated with NCZ. (B) GFP-RPP_139-174-T-agNLS accumulates in the nucleus of transfected cells to a greater extent than GFP-T-agNLS. This increase is inhibitable by NCZ treatment or mutation of D₁₄₃/Q₁₄₇-A. No effect by NCZ is observed on nuclear accumulation of GFP-T-agNLS or GFP-RPP_139-174(D₁₄₃/Q₁₄₇-A)-T-agNLS. (C) CytD treatment does not affect nuclear accumulation of GFP-T-agNLS or GFP-RPP_139-174-T-agNLS. (D) Western blot analysis of transfected cell lysates reveals that all GFP-fused proteins show similar expression levels/stability and NCZ treatment does not affect the RPP_139-174-T-agNLS protein species. (E) RPP_139-151 enhances nuclear localization conferred by T-agNLS to the same levels as RPP_139-174 (GFP-RPP_139-151-T-agNLS and GFP-RPP_139-174-T-agNLS, respectively), and both are equally affected by NCZ treatment. Results are from single assays representative of three or more assays and are shown as mean ± SEM for >60 different cells.