Fig 2. Transient Expression of the three recombinant GARP variants in HEK 293H cells.
HEK 293H cells were transfected with plasmids containing the cDNA of the constructs GARPFL, GARPTS and GARPΔTM, respectively. 48h after transfection, the culture medium was exchanged for FCS-free DMEM supplemented with NEAA. Supernatants (S) and cell lysates (L) were obtained after another 48h of incubation. 1 ml of supernatant was precipitated using 2% (w/v) Na-deoxycholate solution (1:100) and 100% TCA (1:10). 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 α-Strep-tag and α-His-tag antibodies, respectively (Quiagen; Hilden, Germany). As secondary antibody a peroxidase coupled anti-mouse-IgG antibody (Dianova; Hamburg, Germany) was used.