Figure 7. Expression of TDP-43 reduces the binding of VAPB to PTPIP51 in both transfected cells and transgenic mice.

(a,b) Cells were transfected with either empty vector (EV) or co-transfected with PTPIP51-HA and either EGFP control vector (CTRL) or wild-type or ALS/FTD mutants of TDP-43 (TDP-43M337V, TDP-43Q331K, TDP-43A382T and TDP-43G348C). PTPIP51 was immunoprecipitated using the HA tag and the amounts of endogenous bound VAPB detected by immunoblotting. No signals were obtained for either VAPB or PTPIP51 in immunoprecipitations from EV-transfected cells, which demonstrates the specificity of the immunoprecipitations. Both inputs and immunoprecipitations (IP) are shown. The 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. (a) Shows data for NSC34 cells; N=3. (b) Shows data for HEK293 cells; N=5. Data were analysed by one-way analysis of variance and Tukey’s post hoc test; error bars are s.e.m., *P<0.05, **P<0.01. (c) VAPB binds to PTPIP51 in mouse spinal cords. VAPB was immunoprecipitated using rabbit anti-VAPB and detected on immunoblots using rat anti-VAPB; bound PTPIP51 was detected using rat PTPIP51. Both inputs and immunoprecipitations (IP) are shown. Control immunoprecipitations were performed using no primary antibody (−Ab) or preimmune rabbit serum (CTRL). (d) Reduced binding of VAPB to PTPIP51 in TDP-43 transgenic mice. Immunoprecipitations were performed as in c from three non-transgenic (NTg) and three TDP-43 transgenic mice. Also shown are immunoblots for TDP-43, mitofusin-2 (MFN2) and actin as a loading control in the spinal cords. Bar chart shows relative levels of PTPIP51 bound to VAPB in the immunoprecipitations following quantification of signals from immunoblots. PTPIP51 signals were normalized to immunoprecipitated VAPB signals. Data were analysed by the unpaired t-test; error bars are s.e.m., **P<0.01.