Figure 8. ATP13A2 regulates SYT11 levels by additional post-transcriptional processes.

(a) Control and ATP13A2-knockdown HeLa cells were transfected with pEGFP-SYT11 for the last 24 h. Cell lysates were blotted against GFP and actin. (b) HeLa cells were transfected with empty vector or ATP13A2 WT simultaneously with pEGFP-SYT11 for 24 h. Cell lysates were blotted against GFP and actin. (c) Control and ATP13A2-knockdown HeLa cells were transfected with GFP-SYT11 for the last 24 h. In the last 4 h of the experiment, cells were treated with 50 μg ml⁻¹ cycloheximide for the indicated time points. Cell lysates were blotted against GFP and actin. Densitometric quantification of the bands was performed and the normalized data (GFP-SYT11 levels to actin levels) of three independent experiments is plotted in the graph. (d) HeLa cells were transfected with empty vector or ATP13A2 WT simultaneously with HA-Ubiquitin and GFP-SYT11 for 24 h. Cell lysates were subsequently used for GFP-SYT11 immunoprecipitation and western blotting against GFP, ubiquitin and actin. (e) Control and ATP13A2-knockdown HeLa cells were transfected with HA-Ubiquitin and GFP-SYT11 for the last 24 h. Lysates were used for GFP-SYT11 immunoprecipitation and subsequent western blotting against ubiquitin (P4D1 antibody), K48-linkage-specific polyubiquitin conjugates, GFP and actin. (f) Control and ATP13A2-knockdown HeLa cells were transfected with GFP-SYT11 for the last 24 h. In the last 5 h, cells were pre-incubated with 10 μM MG132 (or DMSO) and further treated with 50 μg ml⁻¹ cycloheximide for the indicated time points. Cell lysates were blotted against GFP and actin. Densitometric quantification of the bands was performed and the normalized data (GFP-SYT11 levels to actin levels) of three independent experiments is plotted in the graph. All the graphs represent mean±s.d. and statistical significance was determined using two-tailed paired Student's t-test. *P<0.05; ^**P<0.01.