On-cell ELISA

Cells were seeded in 96 well plates and transfected with 50 ng of one of the following plasmids: pcDNA3.1 (empty vector), P622-CWI, P622-CWA, P622-CAI, and P622-CAA. Twenty-four hours after transfections, cells were starved using DMEM (Thermo Fisher Scientific #21068028) supplemented with glutamine and 1.25 mM Ca²⁺ overnight. Cells were then fixed with 4% paraformaldehyde for 15 min at room temperature. After several washes with TBS, cells were blocked for 30 min in TBSM (TBS + 3% milk) for surface staining or TBSM supplemented with 0.2% Triton X-100 for total staining. Cells were then incubated with either rabbit anti-HA (1:2000) or mouse anti-V5 (1:2000) antibodies in TBSB (TBS + 3% BSA) for 2 h at room temperature. After several washes, cells were incubated for 1 h with 1:2000 dilution of either horseradish peroxidase (HRP)-linked horse anti-mouse IgG (Cell Signaling Technologies #7076) or HRP-linked goat anti-rabbit IgG (Cell Signaling Technologies #7074) antibodies in TBSM. After 5 washes with TBS, cells were incubated with 3,3′,5,5′-Tetramethylbenzidine (TMB) (Sigma #t0440) for 5 min at room temperature. The reaction was stopped by an equal volume of 1 N HCl. Absorbance at 450 nm was measured using a FlexStation III plate reader. In a separate set of experiments, cells were transfected with several amounts of plasmids to determine the doses of mutant plasmids that result in similar surface expression compared to non-mutated P622-CWI or ∆NTF-CWI receptors.