Microsomal proteins from either
D. coccus or yeast synthesizing DcUGT2ΔMD-Strep were deglycosylated using the Enzymatic Deglycosylation Kit for N-linked and Simple O-linked glycans (Prozyme) according to the supplier’s instruction. Deglycosylation was monitored by western blot analysis using either an
anti-Strep antibody (Qiagen, catalog no. 34850; in a dilution of 1:2000) followed by a secondary
HRP-conjugated antibody (Pierce Biotechnology, catalog no. 1858413; in a dilution of 1:5000) or an anti-DcUGT2 antibody (in a 1:1000 dilution) followed by a secondary
HRP-conjugated antibody (Dako, catalog no. P0217; in a dilution of 1:5000) and chemiluminescence detection. Blocking, antibody probing, and washing of the blots were performed according to the manufacturers’ instructions. Uncropped images are shown in Supplementary Fig.
6. The anti-DcUGT2 antibody was obtained by immunizing a rabbit with the sequence-specific peptide, (NH
2)-CEIMFSDPRVLNIRDKKFD-(COOH), representing residues 110–128 in the DcUGT2 protein, conjugated to keyhole limpet hemocyanin (Agisera AB). The pre-immune serum (in a 1:1000 dilution) of the immunized rabbit was tested for cross-reactivity towards a crude
D. coccus protein extract to ensure the anti-DcUGT2 antibody specificity (Supplementary Fig.
7).
Kannangara R., Siukstaite L., Borch-Jensen J., Madsen B., Kongstad K.T., Staerk D., Bennedsen M., Okkels F.T., Rasmussen S.A., Larsen T.O., Frandsen R.J, & Møller B.L. (2017). Characterization of a membrane-bound C-glucosyltransferase responsible for carminic acid biosynthesis in Dactylopius coccus Costa. Nature Communications, 8, 1987.
