|
Fig. 3
THSD7A is an N-glycoprotein and can release the soluble form into the extracellular environment.
A. Empty vector (V)- and THSD7A (T)-transfected HEK293T cells were analyzed by Western blot. THSD7A was detected in both cell lysates and cultured medium. GAPDH served as a cytoplasmic marker. B. Transfected HEK293T cells were incubated in the presence (+) or absence (-) of tunicamycin. After 48 hours, cell lysates and cultured medium were subjected to Western blot and stained with THSD7A-specific and anti-FLAG-tag antibodies (upper panel). The cell lysates and cultured medium of THSD7A-transfected HEH293T cells were treated with (+) or without (-) PNGase and subjected to Western blot (lower panel). C. Cultured medium from transfected HEK293T cells was concentrated by 3 kDa or 100 kDa cutoff Amicon concentrators and analyzed by Western blot. After concentration by a 100 kDa cutoff Amicon concentrator, the 210 kDa soluble THSD7A (arrow) was retained in the concentrates and the 33 kDa amino-terminal fragment of THSD7A (arrowhead) was filtered out.