Biotinylated pna lectin

A Pierce™ Direct IP Kit (Thermo Fisher Scientific) was used according to the manufacturer’s instructions to selectively immunoprecipitate HOMER3 from membrane protein lysates using a rabbit polyclonal anti-HOMER3 antibody (PA5–59383, Thermo Fisher Scientific). The isolated glycoproteins were then run on 4–20% precast SDS-PAGE gels (Bio-Rad), transferred into nitrocellulose membranes and screened for HOMER3 and ST antigens expressions by western blot using the above-mentioned anti-HOMER3 antibody and a biotinylated PNA lectin (Vector laboratories), respectively. ST antigens detection by PNA lectin was preceded by overnight α-neuraminidase from Clostridium perfringens (Sigma-Aldrich) in membrane digestion 0.1 U/mL at 37 °C.

MaxiSorp 96-well plates (Nunc) were coated overnight at 4 °C in carbonate–bicarbonate buffer (pH 9.6) and blocked in PLI-P (PO₄³⁻, Na⁺/K⁺, 1% Triton, and 1% BSA) buffer at pH 7.4 for 1 h at RT. Neuraminidase treatment was performed with 50 mU neuraminidase in 50 mM sodium acetate buffer (pH 5.5) for 1 h at 37 °C. Plates were incubated with mAbs (undiluted culture supernatants), biotinylated PNA lectin (200 ng/ml) (Vector Laboratories), or biotinylated GAL-4 (0.5 μM) for 1 h at RT and followed by washing and incubation with HRP-conjugated antimouse Ig (Dako) or streptavidin–HRP (Dako) for 1 h at RT. Development was started by addition with 3,3′,5,5′-Tetramethylbenzidine substrate (Dako) and stopped with 0.5 M H₂SO₄, and absorbance read at 450 nm after 5 min.