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. 2016 Jun 9;7:11803. doi: 10.1038/ncomms11803

Figure 4. ATP13A2 regulates TSC2 through MYCBP2-induced ubiquitination.

Figure 4

(a) HeLa cells were fixed and analysed by proximity ligation assay (PLA) using primary antibodies against ATP13A2 and MYCBP2. Cells were imaged by epifluorescence microscopy (scale bar, 10 μM). PLA negative controls are shown in the supplements. (b) Endogenous ATP13A2 was immunoprecipitated. Whole-cell lysate and immunoprecipitates were blotted against ATP13A2, MYCBP2, TSC2 and actin. (c) Control and ATP13A2-knockdown HeLa cells were lysed and blotted against phosphoSer1378-TSC2, phosphoThr1462-TSC2, total-TSC2 and actin. (d) Primary cultures of mouse neurons were transduced with Control or ATP13A2 lentiviral shRNAs for 5 days and used for western blotting against TSC2 and actin. (e) HeLa cells were transfected with empty vector, ATP13A2 WT, ATP13A2 delC mutant or ATP13A2 i16 mutant for 24 h. Cells were collected and cell lysates blotted for TSC2 and actin. (f) HeLa cells were transfected with Control or ATP13A2 siRNAs for 4 days. In the last day of transfection, cells were treated with 50 μg ml−1 cycloheximide for the indicated time points (after a pre-treatment of 2 h). Cell lysates were blotted against TSC2 and actin. Densitometric quantification of the bands was performed and the normalized data (TSC2 levels to actin levels) of three independent experiments is plotted in the graph, which represents mean±s.d. *P<0.05 (two-tailed paired Student's t-test). (g) HeLa cells, transfected with two rounds of either Control or ATP13A2 siRNA (50 nM) alone or in combination with MYCBP2 siRNA (50 nM) for 4 days, were further transfected with FLAG-TSC2 and HA-Ubiquitin (1:3 ratio) for 24 h. MG132 (10 μM) was added for the last 5 h. Lysates were used for TSC2 immunoprecipitation. Inputs and immunoprecipitates were blotted against HA, TSC2 and actin. (h) Lysates of HeLa cells transfected with Control or ATP13A2 siRNA alone or in combination with MYCBP2 siRNA (as described before) were blotted against TSC2, phospho p70S6K, total p70S6K, LC3 and actin. The graph represents the mean±s.e.m. of at least three independent experiments. *P<0.05; #P<0.05, ##P<0.01 (two-tailed paired Student's t-test). (i) Control and ATP13A2-knockdown HeLa cells were immunoprecipitated for TSC2. Inputs and immunoprecipitates were blotted against TSC2, MYCBP2 and actin.