Fig 1. Optimization of sample preparation for the identification of binding partners between human TSPO and its mutant A147T by IP-MS.

A) Transfection efficiency of V5-tagged TSPO^WT and TSPO^A147T in U87MG cells (42.6% ± 5.93), indicating equal protein expression levels; red, anti-V5 immunofluorescence and blue, DAPI indicating cell nuclei. n = 3. B) Mitochondrial enrichment of U87MG cells as assessed by Western blot for VDAC as a mitochondrial marker protein and hnRNP as a nuclear marker protein, indicating that purified mitochondria were isolated, n = 2. C) Representative immunoblot of mitochondrial lysates from transfected U87MG cells with V5-tagged TSPO^WT and TSPO^A147T. This confirms equal expression levels of V5 in both TSPO^WT and TSPO^A147T extracts. D) Immunoprecipitation of TSPO^WT and TSPO^A147T from transfected U87MG cells with increasing amount of mouse V5 antibody, with 2μg per 400 μg of total protein chosen for subsequent experiments. Non-specific mouse IgG antibody was used as a negative control. Immunoblot of input (10% of the total lysates) confirms comparable expression levels of V5 tag between TSPO^WT and TSPO^A147T. Immunoblot of unbound V5 tag shows the amount of non-precipitated TSPO. E) Immunoprecipitation of TSPO^WT and TSPO^A147T from transfected U87MG cells. Precipitated proteins were visualized by silver staining of SDS-PAGE.