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. 2017 Mar 23;134(1):129–149. doi: 10.1007/s00401-017-1704-z

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© The Author(s) 2017

Open AccessThis article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made.

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Fig. 8 — α-Synuclein is a MAM protein and binds to VAPB but not PTPIP51 in immunoprecipitation assays. a Immunoblots of total lysates (Lys), MAM, mitochondria (Mit) and ER proteins from rat brains. Samples were probed for α-synuclein plus FACL4, VAPB and calnexin as MAM/ER markers, and PTPIP51 and HSP60 as mitochondrial markers. Extended exposures of immunoblots reveal PTPIP51 signal in total lysate samples. b, c Immunoprecipitation assays of α-synuclein binding to PTPIP51 and VAPB. HEK293 cells were transfected with either PTPIP51-HA (b) or myc-VAPB (c) + either empty control vector (CTRL), α-synuclein, α-synucleinA53T or α-synucleinA30P as indicated. PTPIP51 was immunoprecipitated using rabbit anti-HA and detected on immunoblots with mouse anti-HA; α-synuclein was detected with mouse anti-α-synuclein. VAPB was immunoprecipitated using mouse anti-myc and detected on immunoblots with rabbit anti-HA; α-synuclein was detected with rabbit anti-α-synuclein. Both inputs and immunoprecipitations (IP) are shown. α-Synuclein displayed no binding to PTPIP51 but bound to VAPB. Bar chart in c shows relative levels of α-synuclein bound to VAPB in the immunoprecipitations following quantification of signals from immunoblots. α-Synuclein signals were normalized to immunoprecipitated VAPB-myc signals. Data are expressed as percentage of the wild-type α-synuclein signal and were analysed by one-way ANOVA and Tukey’s post hoc test. N = 4; error bars are SEM, *p > 0.05, **p < 0.01. d, e Endogenous VAPB and α-synuclein bind in immunoprecipitation assays from rat brain. d VAPB was immunoprecipitated using rabbit anti-VAPB and detected with rat anti-VAPB; α-synuclein was detected with mouse anti-α-synuclein. e α-Synuclein was immunoprecipitated using mouse anti-α-synuclein and detected using rabbit anti-α-synuclein; VAPB was detected using rabbit anti-VAPB. Control immunoprecipitations were performed with pre-immune serum or normal mouse Igs. Both inputs and immunoprecipitations (IP) are shown along with immunoglobulin heavy chain signals (Ig) to demonstrate equal Ig amounts in the VAPB and control immunoprecipitations. Molecular masses on immunoblots in kD are shown on the right