(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.