Immunoblotting was performed according
to the guidelines for antibody use in cardiovascular research.49 (link) Validation of glycoproteomic data was performed
by regression analysis comparing mass spectroscopy peak intensity
values to densitometry of protein measured by immunoblotting and normalized
to the intensity of the total membrane stain (Table S1 and Figure S1 ). Samples
were volume-loaded and separated on 4–12% Criterion XT Bis-Tris
gels (Bio-Rad), transferred to a nitrocellulose membrane (Bio-Rad),
and stained with MemCode Reversible Protein Stain Kit (Thermo Scientific)
to verify the protein concentration and loading accuracy. Membranes
were blocked with 5% nonfat milk (Bio-Rad), followed by overnight
incubation with primary antibody [polymeric immunoglobulin receptor
(Abcam ab96196; 1:1000), complement C9 (Abcam ab173302; 1:1000), Apo
F (Raybiotech 102-10552; 1:250), and CD59 (Abcam ab124396; 1:1000)],
followed by secondary antibody (anti-rabbit Vector PI-1000; 1:5000)
and detection with ECL Prime Western Blotting Detection Substrate
(Amersham).
to the guidelines for antibody use in cardiovascular research.49 (link) Validation of glycoproteomic data was performed
by regression analysis comparing mass spectroscopy peak intensity
values to densitometry of protein measured by immunoblotting and normalized
to the intensity of the total membrane stain (
were volume-loaded and separated on 4–12% Criterion XT Bis-Tris
gels (Bio-Rad), transferred to a nitrocellulose membrane (Bio-Rad),
and stained with MemCode Reversible Protein Stain Kit (Thermo Scientific)
to verify the protein concentration and loading accuracy. Membranes
were blocked with 5% nonfat milk (Bio-Rad), followed by overnight
incubation with primary antibody [polymeric immunoglobulin receptor
(Abcam ab96196; 1:1000), complement C9 (Abcam ab173302; 1:1000), Apo
F (Raybiotech 102-10552; 1:250), and CD59 (Abcam ab124396; 1:1000)],
followed by secondary antibody (anti-rabbit Vector PI-1000; 1:5000)
and detection with ECL Prime Western Blotting Detection Substrate
(Amersham).