Western blotting of SCGF after deglycosylation. Western blotting with anti-SCGF-α/-β antibody. Lanes 1 and 2 compare SCGF patterns in the untreated (lane 1) and the deglycosylated (lane 2) protein extract of the GIST 5 sample. Lanes 3-5 contain a comparison of the cytoplasmatic (lane 3) and the secreted (lane 4) SCGF-α forms and the untreated (lane 4) and the deglycosylated secreted (lane 5) SCGF-α forms in the TPC1 cell line. Lanes 6 and 7 depict the expression of SCGF-α and -β in the cell extract and conditioned medium of a mock HEK293 cell line (negative control). Lanes 8-10 reflect SCGF-β expression in the cell extract (lane 8) and conditioned medium (lane 9) of the transfected HEK293 cell line; lanes 8 and 10 show the untreated and the deglycosylated SCGF-β forms in the transfected HEK293 cell line extract. The mature and secreted forms of SCGF-α detected in the GIST 5 sample (lane 1) and the TPC1 conditioned medium (lane 4) are indicated by (a); (b) indicates an immature, cytoplasmatic, unglycosylated form of SCGF-α in the untreated (lane 1) and deglycosylated (lane 2) GIST 5 samples, and in the cell extract (lane 3) and conditioned medium of the TPC1 cell line after deglycosylation (lane 5); the form indicated by (c) was exclusively detected in the GIST 5 sample, was the quantitatively most relevant form in the untreated sample (lane 1), and was observed after deglycosylation (lane 2); (d) indicates the form exclusively detected and quantitatively most relevant in the deglycosylated GIST 5 sample (lane 2), corresponding to the protein backbone of SCGF-α (~36 kDa); (e) indicates the form detected in the untreated (lane 8) and deglycosylated (lane 10) cell extract of the transfected HEK293 cell line, corresponding to the primary structure of SCGF-β (~27 kDa).