Page 280 - 2020_02-Haematologica-web

L. Martorell et al.
and gentamicin; R1960X for geneticin, gentamicin but also PTC124, and p.W2131X for geneticin, and RTC13) or only a minimal qualitative reduction (p.W1726X and p.R2228X) in the intracellular accumulation of truncated FVIII (Online Supplementary Figure S1).
Rescue of FVIII:C activity after readthrough agent treatment
Finally, we focused in the RTA-mediated effect on the protein functionality rather than on the protein levels. To this end, we analyzed the FVIII:C activity by means of a
A
B
Figure 4. Readthrough agents (RTA) reduced intracellular accumulation of FVIII in transfected Chinese hamster ovary (CHO) cells. Representative images of the immunodetection of FVIII heavy chain in cell homogenates of transiently transfected CHO cells. (A) Western blot of CHO cells transfected with either wild-type (WT) BDD-FVIII (166.19 kDa), the p.Q462X (52.8 kDa), p.Q1764X (98.51 kDa) (top panel), p.W2015X (127.9 kDa), or the p.R1822X (105.24 kDa) variants (bottom panel). The intracellular accumulation of BDD-FVIII or the truncated proteins before and after RTA-treatment is shown. (B) Densitometry of the bands in (A). CT: Untreated cells; GN50: 50 μg geneticin/mL; GN100: 100 μg geneticin/mL; GT50: 50 μg gentamicin/mL; GT100: 100 μg gentamicin/mL; PTC: PTC124 10 μM; RTC13 and RTC14: 10 μM; Mock: untransfected cells; MW: Pageruler Plus prestained protein ladder; Refacto®: recombinant human BDD-FVIII (Pfizer); LR: loading reference used for densitometry normalization.
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haematologica | 2020; 105(2)