Measurement of γ-carboxylation of non-haemostatic VKD proteins
The GGCX wt/mut together with VKD proteins in a bicistronic vector were
expressed in a GGCX-/- HEK293T cell line as
previously described (Ghosh et al., 2021). Four hours post transfection
cells were treated with K1. Cell medium and lysate were
collected after 48 hours of transfection. Medium was also collected from
untransfected cells which served as a negative control. Cell medium and
lysates were used to measure γ-carboxylation by a sandwich ELISA.
96-well plates were coated with anti-myc antibody (Applied biological
materials, G077) and were incubated overnight at 4°C. Wells were washed
3x with wash buffer (1x PBS, pH 7.4, 3 mM MgCl2, 0.05%
Tween 20) and blocked in 2% BSA for 2 hours and incubated overnight
with samples in 1:6 dilution. This was followed by 3x washing and 3
hours incubation with anti-Gla antibody. For MGP we used a MGP-specific
Gla antibody (MaGla G8A.1 lot3, gift from Prof. Leon Schurgers). Wells
were then washed 3x and incubated with HRP-conjugated antibody (Dako,
P0260) for 1 hour at RT followed by HRP-dependent chemiluminescence
substrate (Roche, 11582950001) addition and luminescence was detected by
a plate reader (Synergy 2, Biotek).