Fig 4. Verification of GS-YFP transfection and overexpression in human embryonic kidney cells.

Human embryonic kidney cells (HEK293) were transfected with cDNA constructs coding for YFP-tagged human glutamine synthetase (GS-YFP) without (WT) or with mutations at the indicated positions within the GS amino acid sequence (H281A, H284A, Y288A = HHY; S278A, K279A, R280A = SKR). HEK293 cells were either left untreated or were treated with the GS-inhibitor L-methionine-S-sulfoximine (MSO, 3 mmol/l, 2 h). (A): Transfection efficiency was verified by confocal laserscanning microscopy in Hoechst34580 stained cells. (B): Detection of YFP-GS by Western-blot using anti-GFP antibodies. (C): Densitometric quantification of GS-YFP expression levels as detected by anti-GFP antibodies. Anti-GFP immunoreactivity in MSO-treated cells is given relative to the respective untreated control. (D): Detection of GS by Western-blot after heat- and detergent-mediated release of MSO from the enzyme, which restores recognition of GS by the monoclonal anti-GS antibody (BD, clone6). GAPDH served as a loading control. (E): Detection of overexpressed human GS-YFP and endogenously expressed GS by Western-blot using polyclonal anti-GS antibodies (Sigma, Deisenhofen, Germany). GAPDH served as a loading control. * Statisticially significantly different compared to untreated YFP-GS-transfected HEK293 cells (p < 0.05). n.s.: not statistically significantly different.