Assays

2 x 108 wild-type or Tspan15-knockout HEK-293T cells were lysed in 1% digitonin lysis buffer containing protease inhibitor cocktail (Sigma-Aldrich). Lysates were pre-cleared with protein G sepharose prior to immunoprecipitation with Tspan15 mAb 1C12 chemically cross-linked to protein G sepharose with dimethyl pimelimidate (Thermo Fisher Scientific). Five independent immunoprecipitations were carried out for each cell type. Immunoprecipitation samples in non-reducing Laemmli buffer were subjected ...

LUHMES cell culture LUHMES cells were cultured and differentiated as described previously (Sholz et al., 2011). 0.5 Million undifferentiated LUHMES (unLUHMES) cells were seeded into a Poly-D-Lysine coated 6-well containing growth media (DMEM F12, 1% N2 Supplement, 0.04 µg/mL bFGF) and harvested at day three in vitro. The live cell count was around 1 Million cells using Trypan blue and an automated cell counter (Biorad). For differentiation, 1 Million unLUHMES were seeded into a Poly-D-Lysine ...