Figure 2.

Effect of rs704 on vitronectin protein expression, processing, and oligomerization. (A) Western blot analysis of VTN_rs704:C or VTN_rs704:T protein expression after heterologous expression. HEK293 and ARPE-19 cells were transfected with expression vectors for VTN_rs704:C or VTN_rs704:T or with an empty expression vector (pcDNA3.1). Co-transfection with a GFP expression vector was performed as control. Forty-eight and 72 hours after transfection, cell pellets and supernatants (SNs) of transfected cells were subjected to western blot analysis with antibodies against vitronectin and GFP. The ACTB immunoblot served as control. After densitometric quantification, vitronectin signals from cell pellets and supernatants were normalized against GFP. Data represent the mean ± SD of four biological replicates, calibrated against VTN_rs704:C. Asterisks indicate statistically significant differences (^*P < 0.05, Mann–Whitney U test). (B) Non-reducing SDS-PAGE and (C) Blue native PAGE with purified recombinant vitronectin isoforms, followed by western blot analysis against vitronectin.