For detection of MASP3, mouse EDTA plasma (2μl) was resolved on 4–12% Express plus gradient polyacrylamide gel (Genscript #M41212) under non-reducing conditions and transferred to PVDF membranes. MASP3 was detected by sequential incubation with purified rabbit anti-mouse MASP3 IgG (1μg/ml), HRP-conjugated goat anti-rabbit IgG (1:4000 dilution; Sigma-Aldrich) and the ECL chemiluminescent detection system (Amersham Pharmacia Biotech, Uppsala, Sweden). For detection of mouse FD and pro-FD, 2μl Bivalirudin plasma was first deglycosylated with the PNGase F kit from New England Biolabs (cat# P0704S) and proteins were resolved on a 4–20% Express plus gradient polyacrylamide gel (Genscript #M42012) under reducing conditions. FD was detected by using in-house generated rabbit polyclonal anti-mouse FD IgGs (2μg/ml) as the primary antibody and same procedures as in MASP3 detection.
Gullipalli D., Miwa T., Golla M., Sato S., Angampalli S, & Song W.C. (2023). MASP3 deficiency in mice reduces but does not abrogate alternative pathway complement activity due to intrinsic pro-factor D activity. Journal of immunology (Baltimore, Md. : 1950), 210(10), 1543-1551.
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