A9046

Total protein extracts were run on 10% SDS-PAGE and transferred to a nitrocellulose membrane. Membranes were incubated with blocking buffer (5% (w/v) dried milk in TPBS/TBST), rinsed twice in TPBS/TBST and further incubated with blocking buffer supplemented with anti-SigG1 polyclonal quail antibody (final dilution 1:1000) or tetra-his mouse monoclonal antibody (Qiagen), final dilution 1:5000. After incubation, the membranes were rinsed twice in TPBS/TBST, and incubated with secondary antibody—rabbit anti-chicken IgY (IgG) coupled to peroxidase (A9046, Sigma-Aldrich, MI, USA) or anti-mouse IgG conjugated to HRP (Santa Cruz Biotechnology, CA, USA). Signals were revealed with Prime Plus ECL detection kit (Bio-Rad).

Total cell lysates (1.2 × 10⁶ cells) for WB were prepared in SDS–PAGE sample buffer, boiled for 5 min, and sonicated to shear DNA. Proteins were separated using 4 to 20% gradient gels (Mini-PROTEAN TGX; Bio-Rad) and transferred to nitrocellulose membrane (Bio-Rad). After blocking with 10% (vol/vol) adult bovine serum and 0.2% Triton X-100 in PBS for 30 min, the membrane was incubated with chicken anti-BioID2 primary antibody (1:5000; BID2-CP-100; BioFront Technologies). Anti-BioID2 primary antibody was detected using horseradish peroxidase–conjugated anti-chicken (1:40,000; A9046; Sigma-Aldrich) antibody. Biotinylated proteins were detected with horseradish peroxidase-conjugated streptavidin (1:40,000; ab7403; Abcam). The signals from antibodies were detected using enhanced chemiluminescence via a UVP BioImaging System (UVP).

To analyze total cell lysates by immunoblot, 1.2 × 10⁶ cells were lysed in SDS-PAGE sample buffer, boiled for 5 min, and sonicated to shear DNA. Proteins were separated on 4–20% gradient gels (Mini-PROTEAN TGX; Bio-Rad, Hercules, CA, USA) and transferred to nitrocellulose membrane (Bio-Rad). After blocking with 10% (vol/vol) adult bovine serum and 0.2% Triton X-100 in PBS for 30 min, the membrane was incubated with appropriate primary antibodies: chicken anti-BioID2 (1:5000; BID2-CP-100; BioFront Technologies, Tallahassee, FL, USA) or rabbit polyclonal anti-hemagglutinin (1:2000; Ab9110; Lot#GR218331-6, Abcam). The primary antibodies were detected using horseradish peroxidase (HRP)–conjugated anti-chicken (1:40,000; A9046; Lot#015M4856V, Sigma-Aldrich, St. Louis, MO, USA) or anti-rabbit (1:40,000; G21234; Lot#2156243, Thermo Fisher Scientific). The signals from antibodies were detected using enhanced chemiluminescence via a Bio-Rad ChemiDoc MP System (Bio-Rad, Hercules, CA, USA). Following detection of each antibody, the membrane was quenched with 30% H²O² for 30 min. To detect biotinylated proteins, the membrane was incubated with HRP-conjugated streptavidin (1:40,000; ab7403; Lot#GR305788-2, Abcam) in 0.2% Triton X-100 in PBS for 45 min.

IgY concentration was determined by means of an indirect ELISA (enzyme-linked immunoassay) method based on that of Vansofla et al. (2021) with some modifications. We used 96-well plates that were coated with IgG goat
anti-IgY (SAB3700195 Sigma-Aldrich, 2.5  $\mathrm{\mu }$ g m L ${}^{-\mathrm{1}})$ diluted in
bicarbonate buffer (Na ${}_{\mathrm{2}}$ NO ${}_{\mathrm{3}})$ with a pH of 9.6 at 4  ${}^{\circ }$ C overnight.
The, the plates were washed three times using phosphate-buffered saline (PBS)
containing Tween 20 (PBST-20, pH 7.4) and blocked with 100  $\mathrm{\mu }$ L of
2 % bovine serum albumin (BSA) for 1 h at 37  ${}^{\circ }$ C. Serum samples were diluted at a ratio of 1  $:$  100
and were added to each well and incubated for 1 h at 37  ${}^{\circ }$ C.
The plates were washed three times with PBST, and 100  $\mathrm{\mu }$ L of secondary
antibody IgG rabbit anti-IgY (A9046 Sigma-Aldrich) was added to each well
conjugated with a peroxide enzymes. The plate was incubated for 1 h at
37  ${}^{\circ }$ C and then washed three times using PBST. Then 100  $\mathrm{\mu }$ L
of tetramethylbenzidine (TMB) substrate solution (T0440 Sigma-Aldrich) was added to each well.
Finally, the reaction was stopped with stop solution H ${}_{\mathrm{2}}$ SO ${}_{\mathrm{4}}$ , and
the absorbance was read at 450 nm on a microplate reader (Bio-Rad, USA).