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. 2014 Jun 3;5:3996. doi: 10.1038/ncomms4996

Figure 9. TDP-43 activates GSK-3β, and modulating GSK-3β activity regulates the VAPB–PTPIP51 interaction.

Figure 9

(a) Immunoprecipitation assays of TDP-43 with VAPB and PTPIP51. HEK293 cells were transfected with either VAPB (upper) or PTPIP51 (lower) and either control empty vector (EV) or EGFP-tagged TDP-43, TDP-43M337V, TDP-43Q331K, TDP-43A382T or TDP-43G348C. VAPB and PTPIP51 were immunoprecipitated via their myc or HA tags and the samples probed for co-immunoprecipitating TDP-43 on immunoblots. (b) TDP-43 activates GSK-3β. HEK293 cells were transfected with either control empty vector (CTRL), TDP-43, TDP-43M337V, TDP-43Q331K, TDP-43A382T or TDP-43G348C and the samples probed on immunoblots for total GSK-3β and GSK-3β phosphorylated on serine-9. Phosphorylation of GSK-3β serine-9 is the principal mechanism for regulating its activity; serine-9 phosphorylation inhibits GSK-3β activity. Bar chart shows relative levels of GSK-3β serine-9 phosphorylation following quantification of signals from immunoblots and normalization to total GSK-3β signals. Data were analysed by one-way analysis of variance (ANOVA) and Tukey’s post hoc test. N=5; *P<0.05, **P<0.01,***P<0.001; error bars are s.e.m.. (c) Inhibition of GSK-3β increases the amount of VAPB bound to PTPIP51. Cells were transfected with control empty vector (EV) or HA-tagged PTPIP51 and treated with either vehicle, GSK-3β inhibitor AR-A014418 (1 μM) or GSK-3β inhibitor CT99021 (100 nM) for 16 h. PTPIP51 was immunoprecipitated using the HA tag and the amounts of endogenous bound VAPB detected by immunoblotting. Both inputs and immunoprecipitations (IP) are shown. Bar chart shows relative levels of VAPB bound to PTPIP51 in the immunoprecipitations following quantification of signals from immunoblots. VAPB signals were normalized to immunoprecipitated PTPIP51-HA signals. Data were analysed by one-way ANOVA and Tukey’s post hoc test; N=3, error bars are s.e.m., *P<0.05. (d) Transfection of GSK-3β decreases the amount of VAPB bound to PTPIP51. Cells were transfected with empty vector (EV), HA-PTPIP51 or HA-PTPIP51+GSK-3β. PTPIP51 was immunoprecipitated using the HA tag and the amounts of endogenous bound VAPB detected by immunoblotting. Both inputs and immunoprecipitations (IP) are shown. Bar chart shows relative levels of VAPB bound to PTPIP51 in the immunoprecipitations following quantification of signals from immunoblots. VAPB signals were normalized to immunoprecipitated PTPIP51 signals. Data were analysed by the unpaired t-test; N=3, error bars are s.e.m., *P<0.05.