Additional file 2: Figure S2. of Tetraspanin 6: a pivotal protein of the multiple vesicular body determining exosome release and lysosomal degradation of amyloid precursor protein fragments
Creators: name: Guix, Francesc
name: Sannerud, Ragna
name: Berditchevski, Fedor
name: Arranz, Amaia
name: Horré, Katrien
name: Snellinx, An
name: Amantha Thathiah
name: Takaomi Saido
name: Saito, Takashi
name: Sundaresan Rajesh
name: Overduin, Michael
name: Kumar-Singh, Samir
name: Radaelli, Enrico
name: Corthout, Nikky
name: Colombelli, Julien
name: Tosi, Sébastien
name: Munck, Sebastian
name: Salas, Isabel
name: Annaert, Wim
name: Strooper, Bart De
Publisher: name: Figshare
Year: 2017
Subjects: subject: Biophysics
subject: Biochemistry
subject: Cell Biology
subject: Molecular Biology
subject: Neuroscience
subject: 39999 Chemical Sciences not elsewhere classified
subject: FOS: Chemical sciences
subject: FOS: Chemical sciences
subject: Ecology
subject: FOS: Biological sciences
subject: FOS: Biological sciences
subject: Cancer

description: a Microsomal fractions from HEK293 cells downregulating (siRNA-TSPAN6; siRNA-cnt used as control; n = 3 technical repeats per condition) or overexpressing TSPAN6 (TSPAN6; empty vector used as control; n = 3 technical repeats per condition) were prepared for in vitro γ-secretase assay. The γ-secretase inhibitor DAPT (1 μM) was added as a negative control of the reaction. Analysis by western blot of the in vitro assays shows no effects on the production of AICD by downregulating or overexpressing TSPAN6. b In vitro γ-secretase assays were carried out by adding exogenous substrate (3xFlagAPP-C99) to microsomal fractions obtained from HEK293 cells overexpressing TSPAN6 or an empty vector (control) and analyzed by western blot. The γ-secretase inhibitor DAPT (1 μM) was added as a negative control. AICD production (detected with a monoclonal anti-Flag antibody) was not changed by overexpression of TSPAN6. The generation of different Aβ species during the in vitro reactions was determined by ELISA. No statistical differences in the different Aβ species produced were observed. c HEK293 cells treated with siRNA-TSPAN6 or siRNA-cnt were transfected with the γ-secretase substrate myc-NΔE and analyzed by western blot (3 independent experiments). The blot was probed with a monoclonal anti-myc antibody to detect myc-NΔE, a monoclonal antibody against cleaved Notch1 (Val1744), a polyclonal antibody against TSPAN6 and a monoclonal antibody against actin. No effects on the myc-NΔE levels were observed when TSPAN6 is downregulated. d Plot of the ratio calculated from the bands in panel c between the γ-secretase-dependent generation of NICD and its substrate myc-NΔE. Cells down regulating TSPAN6 do not change the production of NICD. e Determination of APP mRNA levels of HEK293 cells transfected with an empty vector (control) or TSPAN6 by qPCR. No significant differences in the APP mRNA levels were observed between control or TSPAN6-overexpressing cells (n = 3 technical repeats per condition). f Determination of BACE1 mRNA levels of HEK293 cells transfected with an empty vector (control) or TSPAN6 by qPCR. No significant differences in the BACE1 mRNA levels were observed between control or TSPAN6-overexpressing cells (n = 3 technical repeats per condition). (PDF 182 kb)