Figure 3: Enhanced HDAC4 protein stability is correlated with greater rod protection efficiency.

(a) Protein decay assay of HDAC4 Full Length and deletion alleles. HEK293T cells were transfected with CAG-HDAC4 or CAG-HDAC4 deletion constructs for 20 h before treatment with cycloheximide (100 μg ml⁻¹) for 3 or 6 h. Cell lysates were immunoblotted for HDAC4. The same immunoblot membranes were stripped and re-probed for actin. (b) Protein levels were quantified by densitometry after normalization to actin. Data are presented as mean±s.d., n=3. (c) Protein decay assay for HDAC4 and HDAC4-S298A/S302A mutant. HEK293T cells were transfected with CAG-HDAC4 or CAG-HDAC4-S298A/S302A for 20 h before treatment with cycloheximide (100 μg ml⁻¹) for 3 or 6 h. Cell lysates were immunoblotted for HDAC4. The same immunoblot membranes were stripped and re-probed for actin. (d) Protein levels were quantified by densitometry after normalization to actin. Data are presented as mean±s.d., n=3. (e) HDAC4-S298A/S302A saved more rd1 rods in comparison to the wild-type HDAC4 at P50. Scale bar, 40 μm. (f) Quantification of the preserved rods per 25,600 μm² on the flat-mount retina. *P<0.01. Data are presented as mean±s.d., n=3. Full scan images of western blot data are provided in Supplementary Fig. 10.