Fig. 3: PAR4 surface expression following PAR4-mRNA-LNP transfection. | npj Vaccines

Fig. 3: PAR4 surface expression following PAR4-mRNA-LNP transfection.

From: To generate functional anti-protease-activated receptor-4 (PAR4), a G protein-coupled receptor, antibodies through PAR4-mRNA-LNP immunization

Fig. 3: PAR4 surface expression following PAR4-mRNA-LNP transfection.

We transfected 0.5, 1, and 2 µg of PAR4-mRNA-LNP into 293 T cells. A To assess the molecular weight of PAR4, an anti-Flag-tag antibody (clone: 9A3) was used to detect Flag-PAR4. In 293T-Flag-PAR4, which served as a positive control, bands were observed at 40 kDa (representing native PAR4 without glycosylation) and 55 kDa (representing native PAR4 with glycosylation). The PAR4-mRNA-LNP transfection groups exhibited a major band at 55 kDa, indicating the presence of the native PAR4 structure with complete glycosylation. The mock group, which did not receive PAR4-mRNA-LNP, served as a negative control. M: Protein ladder, 10–180 kDa. B Surface expression of PAR4 was assessed by flow cytometry using an anti-PAR4 antibody (clone: 5F10, MABS1298). The FITC fluorescence intensity indicated the surface expression of PAR4, which increased in a dose-dependent manner with PAR4-mRNA-LNP transfection. The 293-T cell group acted as a negative control, whereas the 293T-Flag-PAR4 group served as a positive control.

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