Casein blocking buffer

Following SDS-PAGE, proteins were transferred onto nitrocellulose membrane (Bio-Rad) and blocked with 1× Casein Blocking Buffer (Sigma-Aldrich, St. Louis, MO, USA) in PBS for 1 h. Membranes were then incubated on a rocking platform with primary antibodies in 0.5× Casein Blocking Buffer (Sigma-Aldrich) in PBS plus 0.1% Tween-20 (PBS-T) for 1 h. Four different allergen-specific antibodies were utilised: monoclonal anti-tropomyosin (ab50567) (Abcam, Cambridge, UK), in-house generated monoclonal anti-MLC and anti-SBCP raised in mice, and in-house polyclonal anti-HC raised in sheep. For each allergen analysed, 0.5 µg of the respective natural purified shrimp allergen was loaded as a positive control. Bound antibodies were detected with secondary antibodies by using IRDye^® 680RD and IRDye^® 800CW, (Li-Cor Biosciences, Lincoln, NE, USA), and the infrared signals were measured using the Odyssey^® CLx Imaging System (Li-Cor Biosciences). Data from the imaged immunoblots were imported into the Image Studio™ software (version 5.2.5) for analysis of the relative binding intensity. The fluorescence signal of each band was digitised, and the final intensity values were obtained by subtracting the background and blank control.

TalinKO cells were transfected in a 6-well culture plate with either one of two siRNA sequences (Sigma) targeting mouse paxillin (paxillin siRNA 1, 5′-GUC​GUA​AAG​AUU​ACU​UCG​A-3′; paxillin siRNA 2, 5′-CAC​UUU​GUG​UGC​ACC​CAC​U-3′) using Lipofectamine 2000 as per the manufacturers’ instructions. After 48 h, one third of the cells were seeded onto fibronectin-coated glass in the presence of 5 mM Mn²⁺ and fixed 1 h after spreading. Cells were stained for paxillin (rabbit anti-paxillin, GTX125891; GeneTex) and imaged using the Zeiss AxioObserver Z1 wide-field microscope system, as described above, using a 40×/1.3 NA oil objective. Cell area was quantified manually using FIJI.
The remaining cells were lysed using RIPA buffer. 30 µg of protein was applied to SDS-PAGE, and proteins were transferred onto nitrocellulose membranes (Whatman). Membranes were blocked using casein blocking buffer (Sigma) and probed using primary antibodies diluted (1:1,000) in casein blocking buffer. Membranes were washed with Tris-buffered saline (10 mM Tris-HCl, pH 7.4, and 150 mM NaCl) containing 0.05% (vol/vol) Tween 20, followed by incubation with species-specific fluorescent dye–conjugated secondary antibodies (LI-COR Biosciences) diluted in PBS (1:5,000). Membranes were washed again and fluorescent signals were detected using the Odyssey infrared imaging system (LI-COR Biosciences).

Maxisorb plates (Nunc) were coated with anti-CPMV Affimer 3 (concentration and incubation time as shown in Supplementary Fig. 2a) at 4 °C, washed three times and then blocked with 1x casein blocking buffer (Sigma) for 1 hour at room temperature. The plates were washed three times and incubated with 25 µl purified CPMV or leaf extract (diluted in casein blocking buffer to the concentration shown in Supplementary Fig. 2b and Fig. 4) for 1 hour at room temperature. Subsequently the plate was washed three times and incubated with 25 µl of biotinylated cysteine-terminated detection anti-CPMV Affimer 3 (diluted in casein blocking buffer to the concentration shown in Supplementary Fig. 2a) for 1 hour at room temperature, the plates were subsequently washed three times. Bound CPMV and anti-CPMV Affimer were detected by a 1:1000 dilution of HRP-conjugated streptavidin (Pierce) for 1 h at room temperature. The plates were washed six times prior to analysing the binding of CPMV and anti-CPMV Affimer 3 using TMB (Seramun) absorbance at 650 nm.
The optimal sandwich ELISA conditions were determined as; capture anti-CPMV Affimer 3 at 50 µg/ml overnight at 4 °C and detection anti-CPMV Affimer 3 at 0.5 µg/ml.