In vivo coupling of GARP_TS to recombinant TGFβ.

HEK 293H cells were transfected with plasmids containing the cDNA of the constructs GARP_FL, TGFβ_Strep or both in combination. 48h after transfection, the culture medium was exchanged for FCS-free DMEM supplemented with NEAA. Cell lysates were prepared using 200 μl RIPA buffer per 1x10⁶ cells. Samples were separated on a 10% PAA SDS-PAGE followed by western blotting on a PVDF membrane. For molecular size determination the magic mark XP marker (Invitrogen; Darmstadt, Germany) was used. For detection the blot was probed with anti-Strep-tag and anti-His-tag antibodies, respectively (Quiagen; Hilden, Germany). As secondary antibody a peroxidase coupled anti-mouse-IgG antibody (Dianova; Hamburg, Germany) was used.